Promoter Pveg and RBS spoVG for B. subtilis
Constitutive promoter veg( BBa_K143012 ) coupled to the strong Ribosome Binding Site spoVG( BBa_K143021 ) from ''B. subtilis''. Pveg-spoVG can be used in the context of a '''Ribosomes per second''' (RiPS) output generator '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of Pveg was obtained from the DBTBS1 and RBS-spoVG were obtained from papers2 and the sequence synthesised by GeneArt
PliaI promoter of Bacillus subtilis
PliaI is the promoter of the PliaI gene of Bacillus subtilis, which is inducible by bacitracin. It does not contain a ribosome binding site.
Alginate lyase from Bacillus thuringiensis
Alginate lyases destroy the alginate, which is the main component of biofilm in many bacteria, including that produced by Pseudomonas aeruginosa.
As it has never reported the cloning of algynate lyase gene in B. thuringiensis we need to designed the primers using the genome of Bacillus thuringiensis.
We amplified from the genome of Bacillus thuringiensis 4Q7
The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which is able to inhibit the growth of multiples species of plant pathogenic fungi. The RBS used is a strong RBS for Bacillus subtilis (K780002).
This part is composed of a Strong RBS (K780002) and the open reading frame of the Gastrodia anti-fungal protein 1 (GAFP-1) optimized for its expression and its secretion in Bacillus subtilis. The codon optimization was made thanks to the DNA 2.0 software program.
This gene was ordered from a synthesis company.
Multi-host vector pTG262 converted to BioBrick vector wtih LALF protein and SacB signal peptide
Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. The wedge- shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and it has been proposed that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.
This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP through ten amino acid flexible linker sequence. This allows the observation of ''Bacillus subtilis'' DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for ''B.subtilis'' and amp+ for ''E.coli'' and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.
The source of the HBsu coding sequence was the ''B. subtilis'' strain 168 genome, SwissProtTM accession number: P08821. The sfGFP sequence was sourced from BBa_E0040 and the linker from: BBa_K105012.
A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions.
Pspac-hy is a B.subtilis/E.coli promotor inducible by IPTG. It's a hybrid of the phage SPO-1 promotor and lac operator with a point G->T mutation at the -1 site.
pSBBs2E: Empty backbone for integration into Bacillus subtilis lacA locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection in E.coli.
This part is an igem compatible version of the Bacillus subtilis vector pAX01
cotZ: B. subtilis spore crust protein (-2aa)
crust protein See all the details about the LMU-Munich 2012 project here: http://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins
We designed it in Freiburg standard (Assembly 25) and used it for the display of fusion proteins on B. subtilis spores.
The lacI gene encodes a regulator protein for the lactose operon and the hyper-spank promoter of B. subtilis [1].
The part has a Bacillus RBS, but it has no promoter.
The LacI gene was cloned from the B. subtilis shuttle vector pDR111. [2]
This is a ribosome binding site, experimentally verified to work in Bacillus, derived from gfp.
This RBS forms part of the complex device BBa_K104001
EAK16-II is a sixteen amino acid peptide that self-assembles to form β-sheet structures in an aqueous medium. The alternating positive and negative charges (--++--++) are responsible for creating an electrostatic attraction between adjacent peptides 1, triggering self-assembly when the EAK16-II peptides are exposed to physiological media or salt solution. When examined under SEM, a well-ordered nanofibre structure is formed by the association of the EAK16-II peptides and these nanofibres can futher aggregate to form a membranous 3D scaffold. LipA is a signal peptide from the B.subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway2. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by ''B. subtilis''.
BioBrick standard was applied to LipA-EAK16II Fusion Protein.
EAK16-II was identified as a region in zuotin, a Z-DNA binding protein from the yeast genome. LipA originated from the B. subtilis genome3. Both components were produced as a fusion protein by GeneArt.
(Chassis B. subtilis, carrier plasmid pBS0K) Length: 6843 bp Background: pNagA is the promoter of the NagA gene, which expresses a N-acetylglucosamine-6-phosphate deacetylasein in Bacillus subtilis. In bacillus subtilis, this promoter has been reported to induce expression in presence of N-acetylglucosamine 6-phosphate. This NAG molecule is the major component of chitin, a characteristic molecule of fungi. This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
The part includes the pNagA promoter and its RBS (position 3,595,156 to 3,595,356 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping.This part is a subcloning of BBa_K1937003 pNagA part in pSBBS0K_mini plasmid (BBa_K1937001).
The part include the pNagA promoter and its RBS (position 3,595,156 to 3,595,356 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping.This part is a subcloning of BBa_K1937003 pNagA part in pSBBS0K_mini plasmid (BBa_K1937001). In bacillus subtilis, this promoter has been reported to induce expression in presence of N-acetylglucosamine 6-phosphate.
This is the main circuit of our project to allow high expression of target protein(antigen,adjuvant).GFP can be substituted by various proteins via a modular PCR or standard cut/ligation method.
The transdermal-enhancing activity of the TD1 is sequence specific and dose dependent.
"This part was generated in a modified version of RFC25, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGATGGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Stop codons are underlined."
The LMU Bacillus RBS collection is based on a wobble-library with the sequence ARRRRRRGATA and the theoretical translation initiation rates of the RBS in this library, calculated by the salis lab's RBS calculator (https://salis.psu.edu/software/). The 7 RBS submitted as BioBricks are the ones that offer the best coverage of translation initiation rate space, 1 being the optimal Bacillus RBS and 7 being the weakest.
Reflectin 1A (SacB) and LALF (SacB) protein for B.Subtilis
Reflectin is a self-assembling protein that has the ability to reflect the sun light which depends on the thickness of the protein layer. Therefore, we thought that this ability could be used as a novel reporter for B.subtilis and E.coli. On the other hand, Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. The wedge- shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and it has been proposed that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.
LALF from Horseshoe crab, Reflectin from Cephalopod, promoter-RBS from B.subtilis
gfpmut1 (B. subtilis optimized + Freiburg standard)
This gfpmut3 gene was amplified from pGFPamy B. subtilis vector. It is constructed in the Freiburg standard with RBS. prefix: GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC suffix: ACCGGTTAATACTAGTAGCGGCCGCTGCAGT
We designed this part in the Freiburg standard for N- and C-terminal fusions.
This lacZ gene is derived from the Bacillus reporter vector pAC6. It is constructed in the Freiburg Standard (Assembly 25) for in-frame fusion proteins. It also includes a Shine-Dalgarno Sequence optimized for Bacillus subtilis translation.
We constructed the part in the Freiburg standard (assembly 25) for inframe fusion-proteins. There is a Shine-Dalgarno Sequence included between the XbaI and the NgoIV restriction site. It can be used for both N and C terminal fusions.
This BioBrick contains the coding sequence for silk tail and ends with a stop codon. The Tail ....
The DNA sequence was codon-optimized for Bacillus Subtilis. Moreover the nucleotide sequence was optimized in order to accomplish the production standard of IDT. A couple of optimized codons were affected by this.
The amino acid sequence of the tail was obtained from MaSp2. The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
AmyE integratable PoPS generator (Pveg-gsiB) (with CmR)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to a chloramphenicol resistance generator in closed transcriptional unit (Parts BBa_K143052 and BBa_K143064 ) and the PoPS generator Pveg-gsiB ( BBa_K143052 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The chloramphenicol adenyltransferase give resistance to chloramphenicol while the terminator prevents readthrough. The Pveg-gsiB promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of Pveg-gsiB was obtained from papers. Chloramphenicol adenyltransferase and the double terminator were obtained from the registry.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the Pveg-gsiB that was synthesised by GeneArt and Chloramphenicol adenyltransferase and the double terminator that were obtained from the registry.
Epsilon/MazF toxin and antitoxin in pSBBS0K-mini
(Chassis B.subtilis, carrier plasmid pSBBS0K) Length: 6855 bp Background: This Epsilon/MazF part is one of the two components of a double toxin-antitoxin system. The part was designed to be carried on a plasmid while its counterpart MazE/Zeta was carried on another. Unfortunately, only Epsilon/MazF part was successfully cloned in E. coli. This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
Epsilon and MazF are under the control of the promoter Pveg (constitutive promotor for B. subtilis) followed by a strong RBS. A theophylline-dependent riboswitch was used to prevent the MazE toxin expression during the cloning steps in presence of theophylline (Topp and Gallivan, 2008). Indeed, with an expressed toxin, it would be impossible to clone the Biobrick in E. coli.SacII and KpnI are the restriction sites placed respectively before and after the riboswitch so that this fragment can easily be replaced depending on the control we want to apply to the bacteria. The construction was created by integrating the EcorI-PstI fragment from BBa_K1937009 in the pSBBS0K-miniplasmid (BBa_K1937001).
The gene Epsilon codes for a toxin countered by the anti-toxin Zeta (Zielenkiewicz and Cegłowski, 2005 ; Mutschler et al., 2011). The gene MazF encodes the anti-toxin for the MazE toxin (Bravo et al., 1987, Zhang et al., 2005, Wang et al., 2013).
''Bacillus subtilis'' is a the most highly studied gram positive bacterium. In particular, it possess effective secretion systems, is known to be naturally competent for the uptake of DNA and is highly motile. Subspecies 168 is one of the most commonly used in the laboratory and its specific genome has been sequenced.
3??? Integration Sequence for the epsE locus of B. subtilis
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143005) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The EpsE (aka YveO) locus has to our knowledge never been used for integration into ''B.subtilis'' before, but is useful in that it knocks out the potential molecular clutch EpsE gene #1. In particular, both the 5' and 3' integration sequences for the EpsE locus conatin in-frame stop codons to prevent translation of the gene (if nothing is integrated into the locus, integration also prevents correct EpsE expression). The 5' and 3' integration sequences for the EpsE locus were used to integrate over the EpsE gene and prevent its expression in the Imperial 2008 iGEM project ''B.sutbilis'' host. ===References=== #1 pmid=18566286
The EpsE integration sequences were designed from the EpsE (aka YveO) gene's Genbank entry#1 and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu DNA polymerase ===Reference=== #1 http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene, Gene ID:938633
The 3??? integration sequence was taken from the ''B.subtilis'' chromosome and is homologous to the middle section of the EpsE gene. It was produced by PCR cloning with Pfu DNA polymerase
3??? Integration Sequence for the amyE locus of B. subtilis
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into B.subtilis by Shimotsu and Henner[1] and is still commonly used in vectors such as pDR111[2], pDL[3] and their derivatives. Integration at the AmyE locus removes the ability of B.subtilis to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn[4]. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the B.sutbilis chromosome. References 1. Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43(1-2) 85-94. pmid:3019840 2.Nakano S, Kster-Schck E, Grossman AD, and Zuber P. Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis. Proc Natl Acad Sci U S A 2003 Nov 11; 100(23) 13603-8. doi:10.1073/pnas.2235180100 pmid:14597697 3.Bacillus Genetic Stock Center; www.bgsc.org 4.Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74.
The AmyE integration sequence was taken from the vector after comparison by BLAST to the B.subtilis chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 3' end of the gene is the 3' sequence required for integration
The 3??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on B.subtilis, in particular in the studies of transcriptional control[1,2,3] References 1.Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43(1-2) 85-94. pmid:3019840. 2.Erwin KN, Nakano S, and Zuber P. Sulfate-dependent repression of genes that function in organosulfur metabolism in Bacillus subtilis requires Spx. J Bacteriol 2005 Jun; 187(12) 4042-9. doi:10.1128/JB.187.12.4042-4049.2005 pmid:15937167 3.Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 2002 Sep; 184(17) 4881-90. pmid:12169614
Chondroitin 4 sulfotransferase-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 13 amino acids corresponding to amino acids 279 to 290 of the human protein Chondroitin 4 sulfotransferase (UniProt [http://www.uniprot.org/uniprot/Q9NRB3 Q9NRB3]). It has been shown to inhibit NADH oxidase (NOX) mediated binding of ''Streptococcus pneumoniae'' to human A549 cells (Muchnik ''et. al.'', 2013). Displayed on the surface of ''Bacillus subtilis'', it should enable ''B. subtilis'' to bind specifically to ''S. pneumoniae''. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
For generation of the part's nucleotide sequence, the amino acid sequence of the peptide (Muchnik et. al., 2013) was reverse transcribed with regard to the codon usage of Bacillus subtilis. Oligos were designed as precut by EcoRI and PstI to shorten the synthesized fragments and to simplify the cloning into pSB1C3.
The part was generated by annealing of artificially synthesized oligonucletides: CSP_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCGAACCACACATCACTTCCGGCGTCAGCGAGAGAA ACCGGTtaatACTAGTagcggccgCTGCA CSP_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT TTCTCTCGCTGACGCCGGAAGTGATGTGTGGTTCGC CATGCCGGCttcctccttacTCTAGAagcggccgcG
SpoVG ribosome binding site (RBS) for B. subtilis
Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -19kcal 1
In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.
The sequence was taken from a previous research paper [1] and was constructed by Geneart.
In B. subtilis the extracellular matrix is composed of two major components, an EPS and the protein TasA, which polymerizes into amyloid-like fibers.
This is a LacI producing part which is proper for B.subtilis. Any promoter can be added to the downstream of that part to obtain a LacI coding device.
This part is necessary for our kill switch device. To produce LacI in B.subtilis, we designed primers which binds to LacI and have RBS SpoVG sequence as prefix together with RE cut sites.
This part is obtained from BBa_I732820 part by changing the original RBS sequence with RBS SpoVG via specific primers.
There is a G->T point mutation of pSpac at the -1 site, changing the promoter to a stronger one - hence the name pSpac-hy, hy meaning hyper. This promoter drives transcription at rates of about ten times that of the wild type (pSpac) promoter.
Comes from the pPL82 vector, which is intended as a B. subtilis integrational vector. It is placed in front of the MCS in the plasmid, as it would drive the expression of the gene cloned into the MCS both in B. subtilis and E. coli.
SigD-dependent flagellin promoter (Phag) of Bacillus subtilis
Phag promoter of ''B. subtilis'' flagellin under the control of SigD.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. Phag_ENX_fwd: TGCAGAATTCGCGGCCGCTTCTAGAG ttcacaggaaagaaatttacttgacc Phag_SNP_rev: AGCCTGCAGCGGCCGCTACTAGTA gaatatgttgttaaggcacgtcc
2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This part is in the pXyl vector that has been sent to the Igem UNITN 2013 team from the LMU Munich 2012 team. This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. Further information about this part and its characterization can be found in the iGEM Trento 2013 wiki.
===Design Notes=== The coding sequence was taken from Pseudomonas syringae pv. phaseolicola PK2. We firstly optimized the codons for both B. subtilis and E. coli and then synthesized the gene with an RBS sequence upstream of the start codon. We decided to include in the CDS two restriction sites (NgoMIV and AgeI) in order to allow people to use the Freiburg assembly. GAATTCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGCACC... ...TCGACCGGTTAATACTAGTAGCGGCCGCTGCAG
This enzyme was firstly purified from Pseudomonas Siringae pv. phaseolicola PK2, a 2-oxoglutarate-dependent ethylene producing bacterium [1]. The CDS has been optimized for both E.coli and B.subtilis. We decided to include an RBS sequence too.
This part was generated in a modified version of RFC25, and has the following prefix and suffix: {||prefix with EcoRI, NotI, XbaI, SD and NgoMIV:|GAATTCGCGGCCGCTTCTAGATGGCCGGC|-|suffix with AgeI, SpeI, NotI and PstI:|ACCGGTTAATACTAGTAGCGGCCGCTGCAG|}Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Stop codons are underlined.
SigD-dependent promoter of the LytABC locus (PlytA) of Bacillus subtilis
PlytA promoter of the ''B. subtilis'' LytABC operon, under the control of SigD (major), the transcripts being predominants at the exponential growth phase, and SigA (minor).
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. PlytA_ENX_fwd: TGCAGAATTCGCGGCCGCTTCTAGAG ctcatcctttgcacctcg PlytA_SNP_rev: AGCCTGCAGCGGCCGCTACTAGTA tcattatattttatcgtcaacctatt
This part is a sigma factor A-dependent promotor of the gene sdpR derived from the B.subtilis ,and it contains an operon for the signaling pathway of sdpc,a bacterial toxin of B.subtilis. It's activated as the toxin sdpC existed,while the natural pructrue of the promotor, sdpR/I ,confers immunity to the SdpC toxin. Cells of B. subtilis enter the pathway to sporulate under conditions of nutrient limitation could generate sdpC, then this promotor would be activated. We use this part to overexpress sdpC toxin in spore-forming cells,to build a self-killing system in B.subtilis for safety thinking.
from the genome of Bacillus subtilis WB800N
Promoter Pveg, RBS spoVG and SacB signal peptide for B. subtilis
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium.
All three basic components come from B.subtilis genome
Promoter ctc and RBS gsiB for B. subtilis
Sigma B dependant promoter ctc( BBa_K143010 ) coupled to the strong Ribosome Binding Site gsiB( BBa_K143020 ) from ''B. subtilis''. In ''B. subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. Pctc has been used to take an input of blue light and give a '''Ribosomes per second'''(RiPS) output. To work as a blue light receiver correctly, over-expression of the blue light receptor YtvA ( BBa_K143037 ) is required. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter ctc was obtained from the ''B. subtilis'' genome and published papers1 2. RBS-gsiB were obtained from papers3 and the sequence synthesised by GeneArt
The device secrete Rabies virus strain ERA glycoprotein in bacillus subtilis
This device can secrete the Rabies virus strain ERA glycoprotein.it contain the Promoter Pveg, RBS spoVG and SacB signal peptide for B.subtilis (BBa_K541501)and the coding sequence of Rabies virus strain ERA glycoprotein(BBa_K12280002).
Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. The bactericidal activity of lantibiotics is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores. This BioBrick differs in its overhangs from BBa_K1351012.
IPTG-inducible filamentous cell formation
''Bacillus subtilis'' in response to stress such as DNA damage stops the cells from dividing. This is a part of the SOS response initiated by the accumulation of single stranded DNA from DNA damage or stalled replication. Two proteins are vital for this response: RecA and LexA. RecA forms filaments on ssDNA and promotes the autocleavage of LexA. LexA usually represses the SOS operon. ''dinR'' is homologous to lexA in ''E. coli'' and is transcribed in the opposite direction of ''yneA''. ''yneA'' stops the formation of ''ftsZ'' ring indirectly. When ''ftsZ'' forms a 30 subunit ring at the midpoint of the cell, it will contract and cause cell division. By expressing ''yneA'' and inhibiting ''ftsZ'' ring formation, the cells will grow filamentous. By inhibiting cell division, ''yneA'' allows the DNA damage genes to repair the DNA damage before continuing with the cell division cycle. It is hypothesized that ''yneA'' acts through an unknown transmembrane protein to inhibit ''ftsZ'' ring formation; we call this/these unknown components ???Blackbox proteins???. As the evidence shows expression of ''yneA'' leads to filamentation.
Genomic DNA from ''Bacillus subtilis'' -''yneA'' Hyperspankoid from oid and hyperspank
The PcotC gene is the promoter of the CotC gene, and the CotC gene codes for the protein CotC.
Multi-host vector pTG262 converted to BioBrick vector
biobricked version of pTG262 Plasmid has a multi host replication origin and is competant in range of gram positive and gram negative bacteria.
We kindly thank Dr Mike Gasson & Dr. Claire Shearman, of the Institute of Food Research in Norwich for generously donating the pTG262 vector.
SR1 is an untranslated RNA which acts as the antisense to ''ahrC'' mRNA.
The sequence was taken from ''The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism'', Heidrich et al. 2006.
SacB-Human Elastin (EP20-24-24) Fusion Protein
Elastin is a polymeric extracellular matrix protein found in tissues that require the ability to extend and recoil. Examples of elastin containing tissues include arteries, lungs, ligaments and skin. Construct EP20-24-24 for human elastin polypeptide consists of distinct exons which code for alternating hydrophobic regions and crosslinking domains from the human elastin polypeptide gene 1. Under appropriate conditions of temperature and ionic strength, elastin polypeptide undergoes a self-aggregation process known as coacervation. Coacervation is usually induced by an increase in temperature and causes the protein to separate from the solution as a second phase. Unlike most proteins which undergo denaturation when the temperature of the solution increases, elastin polypeptides become more ordered through coacervation 2. SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by ''B. subtilis''. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium. SacB has been successfully used in the secretion of heterologous proteins such as acid-stable α-amylase, cystatin and interleukin-3 by B.subtilis 3.
BioBrick standard was applied to the SacB-Human Elastin(EP20-24-24) Fusion Protein.
All exons in EP20-24-24 are derived from the human elastin polypeptide gene. EP20-24-24 was used to study the effect of various combinations of exons on coacervation of elastin polypeptide. SacB was identified from the initial part of certain preprotein genes that utilises the the Sec-SRP secretory pathway 4. Both components were synthesised as a fusin protein by GeneArt.
Multi-host vector pTG262 converted to BioBrick vector wtih LALF protein and LipA signal peptide
RFP from Discosoma striata (coral) with DAS-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added DAS-ssrA degradation tag (part BBa_M0052). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYADAS and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (AAV in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0052, but same tag was also used in paper by Andersen et al., (1998).[2]
One of three proteins from B. Subtilis which form a membrane transporter that removes subtilin from the membrane, preventing it from forming pores and killing the cells.
Reflectin 1A (SacB) and LALF (LipA) protein for B.subtilis
YtvA is a blue light receptor endogenously found in ''B.subtilis''. Activation of the YtvA light receptor activates the mild stress response pathway, accumulating in sigma B activation. Previous research has shown that sigma B dependent transcription in response to blue light light exposure can be enhanced by the over expression of the YtvA receptor 1. We over expressed YtvA to enhance the transcriptional output of sigma B promoters in response to blue light.
Biobrick standard was applied to the YtvA sequence.
The part was designed using the sequence from the B.subtilis genome. This sequence was then synthesised by Geneart.
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN2). SubunitN2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS.
The amino acids sequence of the silk protein was obtained from MaSp2. The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
RBS - Antifungal D4E1 - Double terminator
D4E1 is a linear synthetic peptide of 17 amino acids which has shown to have antifungal activities by complexing with a sterol present in conidial wall of a variety of fungi. This part is composed of a RBS (K780002 spoVG), the open reading frame of D4E1 and a double terminator (B0015). This part was optimized for the expression and its secretion in Bacillus subtilis.
The coding sequence of D4E1 is flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.
De Lucca AJ,??Bland JM,??Grimm C,??Jacks TJ,??Cary JW,??Jaynes JM,??Cleveland TE,??Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1. Can J Microbiol.??1998 Jun;44(6):514-20.
GsiB ribosome binding site (RBS) for B. subtilis
GsiB is an endogenous ribosome binding site from ''B.subtilis''. The sequence of the gsiB ribosome binding site is '''AAAGGAGG''' which is complementary to the sequence '''UUUCCUCC''' from the 3' region of the 16s rRNA from ''B.subtilis''. GsiB is an endogenous ribosome binding site (RBS) from ''B.subtilis''. The sequence of the gsiB ribosome binding site is '''AAAGGAGG''' which is complementary to the sequence '''UUUCCUCC''' from the 3' region of the 16s rRNA from ''B.subtilis''. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -9.3kcal.
Promoter Pveg and RBS gsiB for B. subtilis
Constitutive promoter veg( BBa_K143012 ) coupled to the strong Ribosome Binding Site gsiB( BBa_K143020 ) from ''B. subtilis''. Pveg-gsiB can be used in the context of a Polymerases per second (PoPS) output generator '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of Pveg was obtained from the DBTBS1 and RBS-gsiB were obtained from papers2 and the sequence synthesised by GeneArt
sleB, Bacillus subtilis germination gene with RBS
''sleB'' encodes for a spore cortex-lytic enzyme. This enzyme responds germination triggered by L-alanine which is the most universal germinant for spores. It has been shown that B. subtilis mutants with inactivated ''sleB'' gene were not able to germinate (1). Hence by regulating this gene in ''sleB'' mutant ''B. subtilis'' cells, germination rate can be contolled.
It has been added as a part with its own natural RBS.
Integration vector for Bacillus subtilis derived from pSac-Cm
This plasmid was derived from pSac-Cm by insertion of biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences, and red fluorescent protein sequence (RFP) in between the prefix and suffix in its multiple cloning sites (MCS). New biobricks can be inserted into this vector by replacement of the RFP biobrick, and selection of the white colonies. This backbone has a multi host replication origin and replicates in E. coli. The plasmid is designed to integrate a cloned insert into the B. subtilis chromosome via double recombination between plasmid and chromosomal sacA sequences, and in order to check for integration a simple Phenol Red Sucrose test can be perform, if sacA is no longer functional, they will remain ph7, if they are still able to digest sucrose their pH will be under 5.
We found that that the integration plasmid that groningen send to the registry were unable to integrate in B. subtilis. This is due to the high number of mutations in the sacA integration region.
Quorum sensing dependant promotor (AIP II). Regulated by phosphorylated AgrA. Contains four AgrA binding sites upstream.
Mutated illegal restriction sites. These sites have been mutated with regard to the codon usage of Bacillus subtilis.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as xylose. XylR if the regulator protein for the Xylose operon in ''B. subtilis''#1 and is responsible for ensuring that in the absence of xylose the xylose metabolism proteins are not expressed. Though endogenous to ''B. subtilis'', to minimise the leakage of a xylose inducible promoter XylR should be over-expressed. In the presence of xylose, the XylR tetramer is unable to bind DNA and so transcription resumes. It must be noted that in all ''B. subtilis'' strains that do not have the Xylose operon knocked out '''the xylose inducer will gradually be metabolised by the host''' XylR was used in conjunction with the '''Xylose operon promoter''' (<bbpart>BBa_K143014<bbpart>) and acted as an input adaptor for a '''Polymerases per second''' (POPS) output
The XylR protein was identified in the genome using its Genbank entry#2 and NCBI's sequence viewer and PCR primers designed from the sequence. Biobrick prefix and suffix sequences were added and the gene cloned by PCR with Pfu DNA polymerase
The XylR protein was PCR cloned form the ''B. subtilis'' genome using Pfu DNA polymerase
Fluorescent ratiometric construct for standardizing promoter output
Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. The construct that uses an inducible eCFP (E0020) and a constitutively expressed eYFP (E0030) under the control of the constitutive promoter Pveg (K143012). The construct is optimized in both E. coli and B. subtilis, through the use of B. subtilis ribosome binding sites GsiB (K143020) and SpoVG (K143021).
This biobrick is best used with a shuttle vector that can function in both E. coli and B. subtilis, which should also have preferably different antibiotic markers in E. coli and B. subtilis.
This biobrick is composed entirely of existing biobricks: GsiB (K143020), eCFP (E0020), terminator (B0015), Pveg + SpoVG (K143053), eYFP (E0030) and a second terminator (B0010). It was constructed first on the E. coli- B. subitilis shuttle vector pJS130, then moved onto pSB1C3 for submission.
This is a Xylose-inducible promoter for use with gram-positive organisms. It requires a downstream gram-positive ribosomal binding site and coding sequence to function. We only have experience using this part in B. subtilis, but it should function in most gram-positive organisms. It has not been tested and will most likely not work in E. coli.
We used the following PCR primers to extract the part, sans biobrick ends: Forward: TTCATGAAAAACTAAAAAAAATATTG Reverse: TATGTCATATTGTAAGTAAGTTGCAC
We PCRed this part out of the pSG1154 gram-positive shuttle vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE153.
RBS Start-codon SpiderSilkSubunitE1 SpiderSilkSubunitE2
This BioBrick contains the coding sequence for two parts of the spider silk proteins (SubunitE1 and SubunitE2) with a Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitE1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites. SubunitE2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS.
The amino acids sequence of the silk protein was obtained from the paper by Brooks et al. (2008). The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
This is a positive/negative selection cassette for Bacillus. If flanked by Bacillus integration sites and integration sites for any chosen DNA, that DNA can be integrated into Bacillus and then the selection cassette can be integrated over by whatever DNA you chose.
***the mazF toxin is unstable in plasmids without the mazE antitoxin***
http://www.bgsc.org/viewdetail.php?bgscid=ECE194&Search=pDGieF http://www.ncbi.nlm.nih.gov/pubmed/16714443
Signal peptide of B. subtilis minor autolysin LytE
Signal peptide (amino acids 1 to 25) of ''Bacillus subtilis'' minor autolysin LytE (probable peptidoglycan endopeptidase, Uniprot [http://www.uniprot.org/uniprot/P54421 P54421]). Targets proteins for secretion via the Sec pathway (Tjalsma ''et. al.'', 2000). This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part was generated by amplification from BioBrick [http://parts.igem.org/Part:BBa_K1351007 BBa_K1351007] with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytE_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGAAAAAGCAAATCATTACAGCTACGACAGC LyteSP_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TGCAGATGCCGCTCCTGCAAATAAC
''Bacillus subtilis'' in response to stress such as DNA damage stops the cells from dividing. This is a part of the SOS response initiated by the accumulation of single stranded DNA from DNA damage or stalled replication. Two proteins are vital for this response: RecA and LexA. RecA forms filaments on ssDNA and promotes the autocleavage of LexA. LexA usually represses the SOS operon. ''dinR'' is homologous to ''lexA'' in ''E. coli'' and is transcribed in the opposite direction of ''yneA''. ''yneA'' stops the formation of ''ftsZ'' ring indirectly. When ''ftsZ'' forms a 30 subunit ring at the midpoint of the cell, it will contract and cause cell division. By expressing ''yneA'' and inhibiting ''ftsZ'' ring formation, the cells will grow filamentous. By inhibiting cell division, yneA allows the DNA damage genes to repair the DNA damage before continuing with the cell division cycle. It is hypothesized that yneA acts through an unknown transmembrane protein to inhibit ftsZ ring formation; we call this/these unknown components ???Blackbox proteins???. As the evidence shows expression of yneA leads to filamentation.
pSBBsOK-mini, a small replicative plasmid for Bacillus subtilis
Part: BBa_K1937001 (pSBBsOK-mini) (Chassis E. coli, carrier plasmid pSB1C3, part destined for use in Bacillus subtilis) Length: 5857 bp This part is an expression vector for Bacillus subtilis. It is replicative both in E.coli and B.subtilis. It has an ampicillin resistance for cloning in E.coli and kanamycin resistance for selection in B. subtilis. This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France) We successfully transformed E. coli and selected clones on ampicillin. Likewise, we successfully transformed B. subtilis and selected clones on kanamycine. The plasmid sequence has been entirely verified by sequencing. The capacity to sub-clone in this new backbone has been demonstrated (part BBa_K1937004, BBa_K1937006, BBa_K1937008 and BBa_K1937010).
The pSBBsOK-mini is a reduction of the pSBBsOK -P plasmid (BBa_K1351040). Its size is only 5827 pb compared to the entire pSBBsOK -P which is 9358 pb long.
The part was created by amplifying a 5827pb fragment from the pSBBsOK -P plasmid (BBa_K1351040) using primers pSBBsOK Forward (5??? ctgctggctagcTACTAGTAGCGGCCGCTGCAGGCGCAACGCAATTAATGTGAGTTAGG) and pSBBsOK Reverse (5??? gacgacgctagcCTCTAGAAGCGGCCGCGAATTcAAATCAGCAAGGGACAGGTAG). The PCR product was digested by NheI and ligated on himself. The resulting plasmid is a lighter shuttle plasmid (E. coli and B. subtilis) with all the properties of the iGEM pSBA13 plasmid (prefix, suffix, verification sequences???).
Verification reverse (VR) primer binding site
Verification forward (VF2) primer binding site
Promoter Pveg, RBS spoVG and LipA signal peptide for B. subtilis
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. LipA is a signal peptide from the B. subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[3]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis.
GFPmut3* has a couple of mutations from wtGFP - S2R and S65G and S72A. Emerald and EGFP have S65T and multiple other mutations. The S2R mutation should have no effect.
1. Elowitz MB and Leibler S. A synthetic oscillatory network of transcriptional regulators. Nature 2000 Jan 20; 403(6767) 335-8. doi:10.1038/35002125 pmid:10659856. PubMed HubMed [Cormack]
GFP gene under expresson of the P43 promoter and spoVG RBS of ''B. subtilis'', with a chloramphenicol resistance marker for ease of selection. Also contains the 5' and 3' amyE integration sequences to allow integration into ''B. subtilis'' at the amyE locus. This device was used by the Imperial iGEM 2008 team to characterise the P43 promoter and spoVG RBS (Combined part BBa_K143051 ) as part of the Biofabricator ''subtilis'' project.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase. The double terminator, GFP gene and chlorapmphenicol acetyltransferase gene were taken from the registry. The sequence of promoter P43 and RBS spoVG were taken from papers.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase and cloned into a BioBrick with the parts from the registry and the P43-spoVG part that was synthesised by GeneArt.
38aa with T1 terminator and Constitutive promoter veg
Human lysozyme (hLZ), the actual protein in breast milk,exhibits microbicidal activity to various degrees against several bacterial strains.The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria.Evidence shows that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria.
38aa doesn't contain any promoter or terminator.
38aa comes from Human lysozyme.It was synthesized in Wuhan Bio-Broad Co.Ltd
This part is a reporter sfGFP(Sp) originally optimized for Streptococcus pneumoniae.
sfGFP originally optimized for streptococcus pneumonia because it could be shown that the signal in Bacillus is even brighter than the one from sfGFP(Bs).
Comprised of araR binding sites and araE promoter. Regulates the expression of downstream genes with arabinose.
Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAGAG-CACAGCGTTCTTTGTAAG (Clamp sequence - Standard Biobrick prefix - first 18 base from the Biobrick) Reverse primer used: TGTGAC-ACTAGTA-GCCCTCCCGAATGTTGAG (Clamp sequence - SpeI site - last 18 base from the Biobrick)
Wild-type Bacillus subtilis 168 strain. 200 bp long bindingsite_promoter_bindingsite_RBS is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5??? and SpeI at 3??? and inserted into a Biobrick compatible vector. The sequence is taken from wildtype Bacillus subtilis
Reflectin 1A (LipA) and LALF (SacB) protein for B.subtilis
LALF from horseshoe crab, Reflectin from Cephalopod and promoter-RBS from B.subtilis
The part is derived from the genomic DNA of B. subtilis 168.
This BioBrick codes for a HBsu protein attached to a red fluorescent protein (RFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the RFP through ten amino acid flexible linker sequence. This allows the observation of ''Bacillus subtilis'' DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for ''B.subtilis'' and amp+ for ''E.coli'' and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.
The source of the HBsu coding sequence was the ''B. subtilis'' strain 168 genome, SwissProtTM accession number: P08821. The RFP sequence was sourced from BBa_E1010 and the linker from: BBa_K105012.
SdpI with RBS: Immunity against the cannibalsim toxin sdpC of B. subtilis
This gene codes for the immunity against a cannibalism toxin of Bacillus subtilis namely the sdpC. The sdpI gene is regulated by a separate promoter (PsdpIR) which is responsible for the operon sdpIR. Cannibalsim toxins are mainly produced by b. subtilis in mid to late stationary phase when nutrients are limited.
No changes were preformed regarding the sequence referring to the bio brick standard.
The sequence comes from a PCR with B. subtilis gDNA template.
This part is the coding sequence for the spaR gene from Bacillus subtilis ATCC6633. SpaR is the response regulator for the subtilin-responsive two component system encoded by the the spaRK operon. Activated SpaR will upregulate promoters that contain a spa-box sequence, such as pspaS.
This promoter forms part of the complex device BBa_K104001
The part comes from the sequence of the spaRK operon from Genbank entry: Bacillus subtilis spaR and spaK genes, complete coding regions, gi|143569|gb|L07785.1|BACSPARK[143569]
A uranyl-binding protein (SUP) with potential uranyl binding sites which are selective for uranyl.
Zhou, L., et al., A protein engineered to bind uranyl selectively and with femtomolar affinity. Nat Chem, 2014. 6(3): p. 236-41.
This is the ribsome binding site for the spaK gene. spaK encodes the sensor kinase for the two component system encoded by the spaRK operon that senses the quorum peptide subtilin.
This part forms part of the complex device BBa_K104001
B. subtilis AmyE locus GFP output promoter and RBS tester
AmyE 3' Integration sequence( BBa_K143002 ) coupled to GFP proein coding region and the registry double terminator (part BBa_I13401 . The amyE 3' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 5' amyE integration sequence( BBa_K143001 ) is cloned onto the 5' end of the construct. The GFP coding region gives a fluorescent output when a promoter and RBS are placed immediately upstream of the coding region while the terminator prevents readthrough.
The amyE 3' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. GFP and the double terminator were obtained from the registry.
The amyE 3' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the GFP and the double terminator that were obtained from the registry.
''Bacillus subtilis'' 168 is unable to biosynthesise flagella. The ''swrA'' gene restores this capability.
''Bacillius subtilis'' 3610 genomic DNA, by PCR.
Several mutated illegal restriction sites. The mutated codons have been optimized regarding the codon usage of Bacillus subtilis.
3??? Integration Sequence for the pyrD locus of B. subtilis
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 3' integration sequence can be added to the back of a Biobrick construct and the 5' integration sequence specific for this locus (BBa_K143008) to the front of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) [1]. Integration into the PyrD locus makes the B.subtilis auxotrophs for uracil and transformants require about 40ug/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negatively select for transformants.
The PyrD integration sequences was designed from the PyrD gene's Genbank entry[1] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined.
The 3??? integration sequence was taken from the B.subtilis chromosome and is homologous to the back section of the PyrD gene. It was synthesised by Geneart.
The promoter PAtpI has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 min of germination [https://www.ncbi.nlm.nih.gov/pubmed/25661487 Sinai''et al.'']. This gene is part of an operon, therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. The region was checked for the binding of sigma factors and transcription factors with DBTBS. No binding factors were found with the highest significance level. In our project we wanted to find a constitutive ON promoter for our ciprofloxacin resistance casette. In the following part we put the promoter PAtpI in the pSB1C3 backbone to make it available to other iGEM teams.
Ordered as gBlock. IDT (Integrated DNA technologies).
GFP gene under expresson of the P43 promoter and gsiB RBS of ''B. subtilis'', with a chloramphenicol resistance marker for ease of selection. Also contains the 5' and 3' amyE integration sequences to allow integration into ''B. subtilis'' at the amyE locus. This device was used by the Imperial iGEM 2008 team to characterise the P43 promoter and gsiB RBS (Combined part BBa_K143050 ) as part of the Biofabricator ''subtilis'' project.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase. The double terminator, GFP gene and chlorapmphenicol acetyltransferase gene were taken from the registry. The sequence of promoter P43 and RBS gsiB were taken from papers.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase and cloned into a BioBrick with the parts from the registry and the P43-gsiB part that was synthesised by GeneArt.
PxylA, a xylose inducible promoter for Bacillus subtilis
P''xylA'' is the native promoter of the ''Bacillus subtilis'' xylose degradation locus ''xyl''. The promoter is regulated by the repressor XylR which is not encoded by this BioBrick. The regulation of P''xylA'' is only ensured, if you are working with ''B. subtilis'' as a chassis.
This is an inducible promoter for Bacillus subtilis.
The part was generated by PCR from genomic DNA of ''B. subtilis'' W168.
CWB domain of B. subtilis minor autolysin LytE
Cell wall binding domain and signal peptide (amino acids 1 to 207) of ''Bacillus subtilis'' minor autolysin LytE (probable peptidoglycan endopeptidase, Uniprot [http://www.uniprot.org/uniprot/P54421 P54421]). This domain has been used for displaying beta-lactamase on the cell surface of ''B. subtilis'' (Chen ''et. al.'', 2008). Used in the [http://2014.igem.org/Team:LMU-Munich/Project BaKillus project] to display pathogen-specific peptides to mediate adhesion of ''B. subtilis'' to pathogens. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
Due to the N-terminal signal peptide, this part allows only C-terminal fusions of proteins to be displayed on the cell surface. The CWB domain of LytE consists of three N‐terminal LysM cell wall‐binding motifs, ranging from amino acid 28 to 193. To minimize possible effects of a translational fusion to the CWB domain, the part includes the downstream unstructered region until amino acid 207. An additional linker can be inserted to allow more structural flexibilty.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytE_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGAAAAAGCAAATCATTACAGCTACGACAGC LytE_207_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT CGATGAAGATGAAGCAGGTTTTGAGC A SpeI site was removed by changing T to C at position 399 without altering the protein sequence by fusion PCR with the following mutagenesis primers: LytE_SpeI399mut_fwd: GTCCTGAAACTGAAAGGTTCAACcAGTTCAAGCAGCTCCAGCTCATCAAAAG LytE_SpeI399mut_rev: CTTTTGATGAGCTGGAGCTGCTTGAACTgGTTGAACCTTTCAGTTTCAGGAC
The PtasA gene is the promoter of the TasA gene, and the TasA gene codes for the protein TasA.
Integrative plasmid for Bacillus subtilis (pSBbs4E)
Promoter gsiB and RBS spoVG for B. subtilis
Sigma B dependant promoter gsiB( BBa_K143011 ) coupled to the strong Ribosome Binding Site spoVG( BBa_K143021 ) from ''B. subtilis''. In ''B. subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. PgsiB has been used to take an input of blue light and give a '''Ribosomes per second'''(RiPS) output. To work as a blue light receiver correctly, over-expression of the blue light receptor '''YtvA''' ( BBa_K143037 ) is required. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter gsiB was obtained from the ''B. subtilis'' genome and published papers1 2. RBS-spoVG were obtained from papers3 and the sequence synthesised by GeneArt
Reflectin1A for B.subtiis with SacB signal sequence
Reflectin is a self-assembling protein that has the ability to reflect the sun light which depends on the thickness of the protein layer. Therefore, we thought that this ability could be used as a novel reporter for B.subtilis and E.coli.
GFP (mut3b) with AAV-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added AAV-ssrA degradation tag (part BBa_I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
GFP comes from part BBa_E0040, tag sequence was obtained from part I11012, but same tag was also used in paper by Andersen et al., (1998).[2]
Natural RBS sequence for Bacillus Subtilis, described in the literature as a strong RBS for this bacteria.
From genomic sequence Bacillus Subtilis.
pSBBs4S-Pxyl: Integrative expression vector for Bacillus subtilis</i
This part is an expression vector for Bacillus subtilis. It integrates at the thrC locus. It has a ampicillin resistance for cloning in E.coli and chloramphenicol resistance for selection in B.subtilis. The multiple cloning site downstream of a Pxyl promoter, that is inducible with xylose. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection for cloning in E.coli.
This part is an igem compatible version of the Bacillus expression vector pXT
Promoter of the liaG gene of Bacillus subtilis, weak constitutive
Promoter xyl and RBS gsiB for B. subtilis
Inducible promoter xyl( BBa_K143014 ) coupled to the strong Ribosome Binding Site gsiB( BBa_K143020 ) from ''B. subtilis''. Pxyl-gsiB can be used to take an input of xylose and give a '''Ribosomes per second''' (RiPS) output generator. Xylose does not directly induce the expression of the promoter xyl, but requires the transcriptional regulator '''XylR''', ( BBa_K143036 ). This means that XylR must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank as an inducible promoter. XylR is naturally expressed by ''B. subtilis'' but should be upregulated to increase efficiency. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter xyl and RBS-gsiB were obtained from papers1 2 3 and the sequence synthesised by GeneArt
This is a dual strong promoter that control the expression of Cry1Ac proteins in Bacillus thuringiensis, but it is expressed constitutively in Escherichia coli. This promoter can control the expression of genes that need to be expressed in a constitutive way in E. coli.
We amplified the promoter from the cry1Ac gene and include restriction sites not found inside the sequence but compatible with the prefix and suffix of the pSB1C3
Promoter Bti-BtII of the cry1Ac gene is found on a megaplasmid of Bacillus thuringiensis subsp. kurstaki HD73
Rachel May Boyd, Phil Hall and Jem Stach
Promoter Hyperspankoid for ''B.subtilis'' adapted pspac-oid and hyperspank
We used pspacoid and the spacing of the repeat from hyperspank.
Combining oid sequence (palindromic) with hyperspank.
Pveg - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator
The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which is able to inhibit the growth of multiples species of plant pathogenic fungi. D4E1 is a linear synthetic peptide of 17 amino acids which has shown to have antifungal activities by complexing with a sterol present in conidia's wall of a variety of fungi. The RBS used is a strong RBS for Bacillus subtilis (K780002).
This part is designed for the co-expression of two different peptides with anti-fungal activities : D4E1 and GAFP-1. It is composed of the strong, constitutive promotor of B. subtilis Pveg (K823003) and the translation unit with GAFP-1 and D4E1 (BBa_K1364012). http://2014.igem.org/wiki/images/4/40/BBa_K1364013.png
This part results from the association of BBa_K823003, BBa_K1364002 and BBa_K1364003.
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE2) and a Strep-tag behind it. SubunitE2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS. The amino acid sequence is based on the adapted MaSp2 sequences discribed in the paper of Brooks et. al. 2008. The Strep-tag, fused at the C terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin.
GFP (mut3b) with SsrA-DAS+2 degradation tag
GFP (mut3b) (see part BBa_E0040) with added engineered NYADAS-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The NYADAS tag encodes peptide sequence AANDENYNYDAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] The two additional residues ???NY??? extends tag between SspB and ClpX binding site, thus preventing clash when both these protein are bound to tag.[3] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [4]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] McGinness, K. E., Baker, T. a & Sauer, R. T. Engineering controllable protein degradation. Mol. Cell 22, 701???7 (2006). [4] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (DAS in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0051.
Gram-positive Shuttle Vector for Chromosomal Integration
This is pBSINT1, a BioBrick-compatible Shuttle Vector that allows for single-copy chromosomal insertion into Bacillus subtilis. It contains the replication origin and Biobrick cut site from pSB1A3 for normal replication in E. coli. It also holds the 5' and 3' flanking regions of the AmyE gene in Bacillus subtilis, and uses these regions of homology to integrate and replace the contents of that locus on the chromosome via a double crossover event. It has two selectable resistance markers, one for Chloramphenicol (5??g/ml) in Bacillus subtiis, and one for Ampicillin (100 ??g/ml) in E. coli. This vector can be used to construct large BioBrick parts using traditional gram-negative-based transformation and minipreps, and then also be used to integrate the completed part into the genome of a gram-positive chassis. As the figure explains, this vector was made via InFusion assembly from several pieces at once. For a detailed protocol of transformation in Bacillus and how to detect integration events at the AmyE locus, see the Cambridge iGEM 2008 wiki protocols page: [http://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation]
We attempted to design the PCR ends to not interfere with the AmyE flanking regions, and also to have the same annealing temperatures. To see the primers we used, see our Primer page under the Bacillus subproject: [http://2008.igem.org/Team:Cambridge/Bacillus/Primers]
As explained in the image, this vector was assembled at once from 4 separate pieces. The green and red regions (biobrick cut site and E. coli replication/Amp resistance) come from PBS1A3. The blue pieces are the flanking AmyE regions from ECE112, which was obtained via the Bacillus Genetic Stock Center.
amyE back; partial alpha-amylase; similar to Genbank U46196
pSBBs1C-lacZ (lacZ reporter vector for B. subtilis)
This part is a backbone vector for Bacillus subtilis. It integrates at the amyE locus. It has a Ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for B.subtilis. The multiple cloning site is followed by a lacZ gene for reporter assays. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This reporter vector can be used with the normal BioBrick assembly strategies.
This vector is the Bacillus vector pAC6 brought into the iGEM BioBrick standard.
Pveg - RBS - Antifungal D4E1 - Double Terminator
This expression cassette is designed for the expression of an antifugal peptide, D4E1, and for its secretion in Bacillus subtilis. D4E1 has shown antifungal activity by complexing with a streol present in condinia's wall of a variety of fungi. It is composed of a constituve promoter (K143012 Pveg), a RBS (K780002 strong RBS), the open reading frame of D4E1 and a double terminator (B0015).
GFP (mut3b) with LAA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LAA-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes E.coli wild type ssrA peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LAA in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E1010, the tag sequence was obtained from part BBa_M0050.
RBS Start-codon EstA Strep-tag SpiderSilkSubunitE1
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE1) with a Bacillus Subtilis ribosome binding site (RBS), a coding sequence for the signal protein EstA and a Strep-tag infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. The EstA codes for a signal protein that that is there to fasilitate secretion of the protein. The Strep-tag, fused at the N terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin. SubunitE1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites. The amino acid sequence is based on the adapted MaSp2 sequences discribed in the paper of Brooks et. al. 2008.
The amino acid sequence of the silk protein was obtained from the paper by Brooks et al. (2008). The DNA sequence was synthesized by Integrated DNA Technologies (IDT).
Biobrick flanked Promoter sequence. The Promoter regulates the sdpI (immunity against the cannibalism toxin sdpC) and the regulator sdpR in bacillus subtilis.
EpsE Molecular Clutch Gene of B. subtilis
The epsE gene of the exopolysaccharide synthesis operon of ''B. subtilis'' has been suggested to function in a manor similar to a molecular clutch#1. If expressed inside a cell it will prevent flagellar movement causing the cell to no longer be able to swim effectively and instead only tumble. As such EpsE could potentially be used as a controller of ''B. subtilis'' movement. Though the EPS operon is normally repressed in ''B. subtilis'', if EpsE is synthetically expressed it would be beneficial for the original copy of epsE to be knocked out. This can be achieved by integrating over the EesE gene with the epsE integration Biobricks ( BBa_K143005 and BBa_K143006 ) which contain 2 in-frame stop codons. Although many bacterial flaggelar assemblies contain proteins that are similar in shape, there is no guarantee that the epsE gene will function correctly in any host cell other than ''B. subtilis''
The epsE(aka yveO) sequence was located in the ''B. subtilis'' chromosome#1 and the ''Pst''I restriction site removed before synthesis by GeneArt
The epsE gene sequence was taken from the ''B. subtilis'' chromosome and was synthesised by GeneArt.
Laminin-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 9 amino acids corresponding to amino acids 661 to 668 of the human protein Laminin (subunit alpha-5, UniProt [http://www.uniprot.org/uniprot/O15230 O15230]). It has been shown to bind to NADH oxidase (NOX) of ''Streptococcus pneumoniae'' and inhibit bacterial binding to human A549 cells (Muchnik ''et. al.'', 2013). Displayed on the surface of ''Bacillus subtilis'', it should enable ''B. subtilis'' to bind specifically to ''S. pneumoniae''. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
The part was generated by annealing of artificially synthesized oligonucletides: L5P_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG TGTCAAGAATGTTCACCGGGATTT ACCGGTtaatACTAGTagcggccgCTGCA L5P_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT AAATCCCGGTGAACATTCTTGACA CATGCCGGCttcctccttacTCTAGAagcggccgcG
BioBrick-compatible ''rocF'' coding sequence, which codes for the enzyme arginase.
Single base mutation to remove EcoR1 site
Promoter veg a constitutive promoter for B. subtilis
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
Cell wall-anchoring domain of B. subtilis endonuclease YhcR
Cell wall-anchoring domain (amino acids 1086 to 1217) of ''Bacillus subtilis'' endonuclease YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]). YhcR is secreted and covalently anchored to the petidoglycan wall by the sortase YhcS. A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the ''B. subtilis'' cell wall (Liew ''et. al.'', 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of ''B. subtlis'' to pathogens. It consists of four components: the sortase substrate with a signal peptide ([http://parts.igem.org/Part:BBa_K1351008 BBa_K1351008]), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain (this part) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 BBa_K1351011]), whose overexpression increases the efficiency of the surface display. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. CWAD_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCTGAAGCCAGTGATATTGAAGACCC CWAD_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TCACGTTCTGGAGGCGCTC
pSB1AC3-HySp: Integrational backbone into B. subtilis amyE locus with IPTG inducible promoter
We used the amyE intergrational backbone form Munich 2012 (BBa_K823023) that had no promoter. We've added the Hy_Spank IPTG inducable promoter from Rudner's Lab 2004. The Hy_Spank promoter is right in front of the prefix so any biobrick can be inserted in this backbone.
In pDR111 the Hy_spank promoter and the lacI gene are orientated in the same direction. In order to have the Hy_spank promoter right in front of the prefix we turend the promoter around.
iGEM Munich 2012 (Rudner Lab 2004) and Rudner's Lab 2004 (pDR111)
Strong constitutive promoter for Bacillus subtilis
'host-takover module' from B.subtilis Bacteriophage SPO1
Encryption key sequence in integration plasmid (BBa_K823023)
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The key sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted key sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our key sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
Key sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
Rachel May Boyd, Phil Hall, Deena Tsu, Jannetta Steyn
''Bacillus subtilis'' in response to stress such as DNA damage stops the cells from dividing. This is a part of the SOS response initiated by the accumulation of single stranded DNA from DNA damage or stalled replication. Two proteins are vital for this response: RecA and LexA. RecA forms filaments on ssDNA and promotes the autocleavage of LexA. LexA usually represses the SOS operon. ''dinR'' is homologous to ''lexA'' in E. coli and is transcribed in the opposite direction of yneA. yneA stops the formation of ''ftsZ'' ring indirectly. When ''ftsZ'' forms a 30 subunit ring at the midpoint of the cell, it will contract and cause cell division. By expressing ''yneA'' and inhibiting ''ftsZ'' ring formation, the cells will grow filamentous. By inhibiting cell division, yneA allows the DNA damage genes to repair the DNA damage before continuing with the cell division cycle. It is hypothesized that yneA acts through an unknown transmembrane protein to inhibit ''ftsZ'' ring formation; we call this/these unknown components ???Blackbox proteins???. As the evidence shows expression of ''yneA'' leads to filamentation.
QnrS1 resistance cassette BioBrick BBa_K1930004.
To design a QnrS1 resistance cassette BioBrick BBa_K1930004 we designed a gBlock that contains the Bacillus subtilis promoter PAtpI, which is active from a very early stage of germination and includes a ribosome binding site. The gBlock also contains the original QnrS1 gene sequence from E. coli, the double terminator BBa_B0015 from iGEM as well as the prefix and suffix for BioBricks. In summary the ciprofloxacin cassette consists of the following parts PAtpI+RBS+QnrS1+2TER.
This part encodes a two-component system that can be used to sense the level of a small lantibiotic peptide called subtilin. It belongs to a series of parts that can be used to sense quorum sensing peptides from Gram positive organisms. In the Newcastle 2008 iGEM entry it was used a 'proof of concept' brick to test whether a peptide produced by one organism could be sensed by a different organism. The part contains two orfs arranged in an operon, SpaK and SpaR, that are driven by their native RBSs and the promoter PspaRK. Downstream of the SpaRK operon is terminator followed by a RBS and a promoter PspaS. In response to subtilin, the sensor kinase SpaK will phosphorylate, and hence activate, the response regulator SpaR. The phosphorylated SpaR protein activates the PspaS promoter driving the expression of any downstream genes. In this brick there are no ORF's downstream of PspaS but the brick has been tested with gfp and mcherry by the Newcastle 2008 team.
A terminator was included after the spaRK to prevent read through. The native SpaRK promoter is controlled by the sigma factor sigma-H and induced at the end of exponential phase. The regulation by sigma-H was left unmodified.
The two component system, its promoter and RBS's, and the pSpaS promoter sequence are all derived from the genome of Bacillus subtilis ATCC 6633.
Pveg-spoVG + EcAMP-1 + Double terminator
This is the consensus RBS for Bacillus subtilis.
The part must be 8 nucleotides away from the first methionine of the coding sequence, and so we added 'TGT' downstream of the actual binding sequence: AAAGGAGG. This, combined with the truncated non-CDS BioBrick prefix scar, gives exactly 8 'spacer bases' between the 3' end of the RBS and the start of the coding sequence.
PcomAB, a CSP inducible promoter derived from Streptococcus pneumoniae
CSP-depending promoter of Streptococcus pneumoniae. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCCGCGGCCGCTTCTAGATAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
RFP gene under expresson of the P43 promoter and gsiB RBS of ''B. subtilis'', with a chloramphenicol resistance marker for ease of selection. Also contains the 5' and 3' amyE integration sequences to allow integration into ''B. subtilis'' at the amyE locus. This device was used by the Imperial iGEM 2008 team to characterise the P43 promoter and gsiB RBS (Combined part BBa_K143050 ) as part of the Biofabricator ''subtilis'' project.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase. The double terminator, RFP gene and chlorapmphenicol acetyltransferase gene were taken from the registry. The sequence of promoter P43 and RBS gsiB were taken from papers.
RFP gene under expresson of the Pveg promoter and spoVG RBS of ''B. subtilis'', with a chloramphenicol resistance marker for ease of selection. Also contains the 5' and 3' amyE integration sequences to allow integration into ''B. subtilis'' at the amyE locus. This device was used by the Imperial iGEM 2008 team to characterise the Pveg promoter and spoVG RBS (Combined part BBa_K143053 ) as part of the Biofabricator ''subtilis'' project.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase. The double terminator, RFP gene and chlorapmphenicol acetyltransferase gene were taken from the registry. The sequence of promoter Pveg and RBS spoVG were taken from papers.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase and cloned into a BioBrick with the parts from the registry and the Pveg-spoVG part that was synthesised by GeneArt.
Promoter 43 and RBS spoVG for B. subtilis
Constitutive promoter 43( BBa_K143013 ) coupled to the strong Ribosome Binding Site spoVG( BBa_K143021 ) from ''B. subtilis''. P43-spoVG can be used in the context of a Polymerase per second (PoPS) output generator. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of P43 and RBS-spoVG were obtained from papers1 2 and the sequence synthesised by GeneArt.
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE1) with Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitE1 codes for a spider silk protein that is optimized to prevent the forming of secundary RNA structures in close proximity to the RBS. This is given priority over reducing the number of restriction sites. However, the primary goal of the algorithm was still to optimize the codon selection based on their availbility scores.
Promoter hyper-spank and RBS gsiB for B. subtilis
Inducible promoter hyper-spank( BBa_K143015 ) coupled to the strong Ribosome Binding Site gsiB( BBa_K143020 ) from ''B. subtilis''. Phyperspank-gsiB can be used to take an input of IPTG and give a '''Ribosomes per second''' (RiPS) output generator. IPTG does not directly induce the expression of the promoter hyper-spank, but requires the transcriptional regulator '''LacI''', ( BBa_K143035 ). This means that LacI must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank as an inducible promoter. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter hyperspank was obtained from the ''B. subtilis'' integration vector pDR111 and RBS-gsiB were obtained from papers1 and the sequence synthesised by GeneArt
Phyperspank-gsiB was synthesised by GeneArt
Bacillus subtilis ars promoter and arsR gene plus E. coli lacZ
This part includes the arsenate-responsive ars promoter from Bacillus subtilis (Sato, T., and Kobayashi, Y. 1998. J. Bacteriol. 180, 1655-1661), together with the arsR gene encoding the negatively autoregulated repressor ArsR, plus the Escherichia coli lacZ' gene encoding the N-terminal 137 amino acid residues of LacZ, sufficient to complement the chromosomal lacZ-delta-M15 mutation carried by many common lab strains of E. coli. This construct is designed to give LacZ' expression in the presence of arsenic as arsenate or arsenite anion. In previous experiments using other reporter genes such as xylE (available as biobrick BBa_J33204), we have found that this promoter gives a more rapid and stronger response than the E. coli chromosomal ars promoter (available as biobrick BBa_J33201, and fused to lacZ' as BBa_J33203), but with higher background activity in the absence of arsenate. Our experiments with this particular construct showed no obvious inducibility of a pH response due to a high background expression level in the absence of arsenate; however, ONPG assays have not yet been done. This construct does not include a terminator, so another reporter gene could be added at the 3' end for further experiments.
The B. subtilis ars promoter and arsR gene were amplified as a biobrick-like component, but unfortunately a base of the biobrick suffix was inadvertently omitted, so that this could not be submitted alone as a proper biobrick. The lacZ' component is similar to that submitted as BBa_J33202, but differs in including a larger amount of DNA, encoding 137 amino acid residues rather than 76. This was isolated as an unexpected minor product from the PCR reactions which gave BBa_J33202. It appears to complement the lacZ-delta-M15 mutation equally well. The two components were joined as biobricks to generate the construct given here.
The B. subtilis ars promoter and arsR gene were amplified from B. subtilis 168 genomic DNA using primers based on the published genome sequence. The E. coli lacZ' was amplified from E. coli BL21 genomic DNA using primers based on published sequence data (Genbank accession J01636, gi:146575).
Anderson promoter J23107 (with one nucleotide different to the original Anderson promoter sequence) without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
AmyE integratable PoPS generator (Pveg-spoVG)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to the PoPS generator Pveg-spoVG ( BBa_K143053 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The Pveg-spoVG promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of Pveg-spoVG was obtained from papers.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the Pveg-spoVG that was synthesised by GeneArt.
B. subtilis AmyE locus RFP output promoter and RBS tester
Long: AmyE 3' Integration sequence( BBa_K143002 ) coupled to RFP proein coding region and the registry double terminator (part BBa_J04650 . The amyE 3' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 5' amyE integration sequence( BBa_K143001 ) is cloned onto the 5' end of the construct. The RFP coding region gives a fluorescent output when a promoter and RBS are placed immediately upstream of the coding region while the terminator prevents readthrough.
The amyE 3' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. RFP and the double terminator were obtained from the registry.
The amyE 3' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the RFP and the double terminator that were obtained from the registry.
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN2) and a Strep-tag behind it. SubunitN2 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to minimize the number of restriction sites, and the tetrary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS. The Strep-tag, fused at the C terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin.
Xylose operon repressor protein - Terminator
Xylose operon repressor protein(XylR)( BBa_K143036 ) coupled to the double terminator ( BBa_B0015 . XylR can be used in conjunction with the xylose operon promoter ( BBa_K143014 ), where the XylR will act as a receiver for a xylose input to result in a '''Polymerases per second''' (PoPS) output. The double terminator is the most commonly used terminator and is a combination of parts BBa_B0010 and BBa_B0012 . The double terminator allows the XylR to be easily incorporated into a closed transcriptional unit.
XylR was identified from the ''B. subtilis'' chromosome. The double terminator is the most commonly used registry terminator.
XylR was produced by PCR cloning using Pfu form the ''B. subtilis'' chromosome and cloned into a BioBrick with the registry double terminator
Signal peptide of B. subtilis endonuclease YhcR
Signal peptide (amino acids 1 to 46) of ''Bacillus subtilis'' endonuclease YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]. YhcR is secreted and covalently anchored to the petidoglycan wall by the sortase YhcS. Despite its signal protein of RR-type, which is typical for proteins exported via the Tat pathway, export of YhcR is not Tat-dependent and seems to function via the Sec pathway (Tjalsma ''et. al.'', 2004). A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the ''B. subtilis'' cell wall (Liew ''et. al.'', 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of ''B. subtlis'' to pathogens. It consists of four components: the sortase substrate with a signal peptide (this part), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain ([http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 BBa_K1351011]), whose overexpression increases the efficiency of the surface display.
The native signal peptide of YhcR was used instead of known Sec-dependent signal peptides as Liew ''et. al.'' did 2012.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. SP_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGCTGTCTGTCGAAATGATAAGC SP_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT AGCTTCGAACGTGTACATTACATTTAAG
Our terminator is based on strong, Rho-independent terminator of gyrase gene, which is type II topoisomerase (EC 5.99.1.3). DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils or relaxes positive supercoils. Negative DNA supercoiling is essential in vivo to compact the genome, relieve torsional strain during replication, and promote local melting for vital processes such as transcript initation by RNA polymerase. Gyrase gene is an housekeeping gene and because of that it must have efficient terminator.
Terminator of gyrase (EC 5.99.1.3) gene.
AmyE integratable PoPS generator (P43-gsiB)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to the PoPS generator P43-gsiB ( BBa_K143050 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The P43-gsiB promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of P43-gsiB was obtained from papers.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the P43-gsiB that was synthesised by GeneArt.
5??? Integration Sequence for the amyE locus of B. subtilis
The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into ''B.subtilis'' by Shimotsu and Henner#1 and is still commonly used in vectors such as pDR111#2, pDL#3 and their derivatives. Integration at the AmyE locus removes the ability of ''B.subtilis'' to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn#4. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the ''B.sutbilis'' chromosome. #1 pmid=3019840 #2 pmid=14597697 #3 ''Bacillus'' Genetic Stock Center [www.bgsc.org] #4 Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74.
The AmyE integration sequence was taken from the vector after comparison by BLAST to the ''B.subtilis'' chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 5' end of the gene is the 5' sequence required for integration.
The 5??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on ''B.subtilis'', in particular in the studies of transcriptional control#1 #2 #3 #1 pmid=14597697 #2 pmid=15937167 #3 pmid=12169614
Gram-Positive General Constitutive Promoter
This is a constitutive (always-on) promoter for use with gram-positive organisms. It requires a downstream gram-positive ribosomal binding site and coding sequence to function. We only have experience using this part in B. subtilis, but it should function in most gram-positive organisms. It has not been tested and will most likely not work in E. coli.
We found the functional annotation in the associated paper: Kaltwasser M., T. Wiegert, and W. Schumann. 2002. Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis. Appl. Environ. Microbiol 68:2624-2628 The primers we used to extract it (sans biobrick ends) are as follows: Forward: AAACGAGGTCATCATTTCCTT Reverse: CAAATGTAGTCTTTGAAAGTATTACA
We PCRed this part out of the pMUTIN-YFP gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE151.
Monomeric Red Fluorescent Protein from Discosoma striata
Mutant of the Biobrick BBa_E1010, in order to make it compatible with the Freiburg-Standard, two AgeI-Restriction sites were deleted monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
The Biobrick has the Following Prefix and Suffix: Gatcgaattccgcggccgcttctagataaggaggagccggc Gatcctgcagcggccgctactagtattaaccggt
YtvA light receptor Protein - Terminator
YtvA light receptor protein (YtvA)( BBa_K143037 ) coupled to the double terminator ( BBa_B0015 . YtvA can be used in conjunction with a sigma factor B specific promoter ( BBa_K143010 or BBa_K143011 ), where the YtvA will act as a receiver for a blue light input to result in a '''Polymerases per second''' (PoPS) output. The double terminator is the most commonly used terminator and is a combination of parts BBa_B0010 and BBa_B0012 . The double terminator allows the YtvA to be easily incorporated into a closed transcriptional unit.
YtvA was identified from the ''B. subtilis'' chromosome. The double terminator is the most commonly used registry terminator.
YtvA was synthesised by GeneArt and cloned into a BioBrick with the registry double terminator.
Gold-specific binding protein (GolB) is a gold-binding chaperon discovered in S. Typhimurium which has been verified that the display of GolB on E.coli cell surface permitted selective enrichment of gold ions from media containing various metal ions.
Encrypted message sequence in integration plasmid (BBa_K823023)
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation. For more information see plasmid construction under our lab journal: http://2016.igem.org/Team:Groningen/Experiments
Message sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
Pveg - strong RBS - Antifungal GAFP-1 - Double terminator
The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which is able to inhibit the growth of multiples species of plant pathogenic fungi.
This part is composed of the strong, constitutive promoter of Bacillus subtilis Pveg (K823003), strong RBS (K780002), the open reading frame of the Gastrodia anti-fungal protein 1 (GAFP-1) and a double terminator B0015. optimized for its expression and its secretion in Bacillus subtilis. The codon optimization was made thanks to the DNA 2.0 software program.
This part results from the association of BBa_K143012, BBa_K1364002 and BBa_B0015.
One of three proteins from ''B. Subtilis'' which form a membrane transporter that removes subtilin from the membrane, preventing it from forming pores and killing the cells.
Overlaps with ''spaI'' to the 5' and ''spaE'' to the 3'
AmyE integratable PoPS generator (Pveg-spoVG) (with CmR)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to a chloramphenicol resistance generator in closed transcriptional unit (Parts BBa_K143053 and BBa_K143064 ) and the PoPS generator Pveg-spoVG ( BBa_K143053 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The chloramphenicol adenyltransferase give resistance to chloramphenicol while the terminator prevents readthrough. The Pveg-spoVG promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of Pveg-spoVG was obtained from papers. Chloramphenicol adenyltransferase and the double terminator were obtained from the registry.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the Pveg-spoVG that was synthesised by GeneArt and Chloramphenicol adenyltransferase and the double terminator that were obtained from the registry.
''Bacillus subtilis'' 168 is unable to produce surfactin, a natural surfactant which helps in reducing surface tension and aids bacteria to swarm on a solid or semi solid medium. The ''sfp'' gene restores the capability to produce surfactin.
''Bacillus subtilis'' 3610 genomic DNA, by PCR.
This is the sigma H type promoter that normally drives the expression of the spaRK operon in Bacillus subtilis ATCC6633. SigmaH promoters are normally active at the early onset of stationary phase growth.
Pveg + Chitin Binding Protein for Bacillus subtilis
pSBBs1C: Empty backbone for integration into Bacillus subtilis amyE locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the amyE locus and can be selected with Chloramphenicol (cat gene). It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1662. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR.
This backbone is an igem compatible version of the Bacillus vector pDG1662
Multi-host replicative plasmid, for B. Subtilis and E. Coli
This plasmid is an multihost plasmid, replicated both in E. Coli at high copy number (>200) and low copy in B. Subtilis (about 20). This plasmid is ideal for importing parts in Subtilis: all the cloning can be done in E. Coli, as well as the amplification. Once the plasmid is extracted, you can incorporate it directly in B. Subtilis using electroporation. This plasmid is Ampicilyn resistant in E. Coli, and is tetracycline resistant in both B. Subtilis and E. Coli. The biobrick BBa_J04450 is inserted between the E X and SP site, has a negative cloning control in E. Coli
The EcoRI site has to be eliminated and the Biobricks prefixes and suffixes were recovered
The plasmid was obtained from Harald Putzer laboratory at the IBPC [http://www.ibpc.fr/UPR9073/equipe_HaraldJackie/AccueilJAP_HPGB.htm] . This is the fusion of the commercial plasmids pBC16-1 and pTZ19R via the site EcoRI. This site has been eliminated by the 2011 iGEM Paris Team by quick change mutagenesis, and the multicloning site was transformed into the biobrick standards.
RBS Start-codon EstA Strep-tag SpiderSilkSubunitN1
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN1) with a Bacillus Subtilis ribosome binding site (RBS), a coding sequence for the signal protein EstA and a Strep-tag infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. The EstA codes for a signal protein that that is there to fasilitate secretion of the protein. The Strep-tag, fused at the N terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin. SubunitN1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites.
RBS Start-codon Strep-tag SpiderSilkSubunitE1
The part contains the coding sequence for part of the spider silk protein together with Bacillus Subtilis ribosome binding site (RBS) and the Strep-tag.
Contactin-4-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 13 amino acids corresponding to amino acids of the human protein Contactin-4 (isoform 3, UniProt Q8IWV2-3). It has been shown to bind to NADH oxidase (NOX) of Streptococcus pneumoniae and inhibit bacterial binding to human A549 cells (Muchnik et. al., 2013). Displayed on the surface of Bacillus subtilis, it should enable B. subtilis to bind specifically to S. pneumoniae. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
The part was generated by annealing of artificially synthesized oligonucletides: C4P_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG TGGGAATGTAAAGCGAACGGAAGACCGAAACCGACATAT ACCGGTtaatACTAGTagcggccgCTGCA C4P_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT ATATGTCGGTTTCGGTCTTCCGTTCGCTTTACATTCCCACATGCCGGCttcctccttacTCTAGAagcggccgcG
Optimized for both E.coli and B.subtilis
5??? Integration Sequence for the pyrD locus of B. subtilis
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus ( BBa_K143004 ) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) 1. Integration into the PyrD locus makes the ''B.subtilis'' auxotrophs for uracil and transformants require about 40ug/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negatively select for transformants.
The PyrD integration sequences were designed from the PyrD gene's Genbank entry#1 and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and PyrD gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined.
The 5??? integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosome from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. The part was synthesised by Geneart.
B. subtilis Episomal Vector with Constitutive GFP
This episomal vector has within its BioBrick region a constitutively expressed GFP gene. It uses the pUpp strong constitutive promoter and a consensus Bacillus RBS sequence.
To build this part, we used 4 different linear DNA pieces assembled with 15 base pair homologous regions PCRed to the ends. These 4 different pieces were then assembled via the InFusion assembly method by Clontech.
See data for BBa_K90401. This part is the same, but with the pUpp promoter, a gram-positive RBS, and a GFP with terminator.
EAK16-II is a sixteen amino acid peptide that self-assembles to form β-sheet structures in an aqueous medium. The alternating positive and negative charges (--++--++) are responsible for creating an electrostatic attraction between adjacent peptides 1, triggering self-assembly when the EAK16-II peptides are exposed to physiological media or salt solution. When examined under SEM, a well-ordered nanofibre structure is formed by the association of the EAK16-II peptides and these nanofibres can futher aggregate to form a membranous 3D scaffold. SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by ''B. subtilis''. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium. SacB has been successfully used in the secretion of heterologous proteins such as acid-stable α-amylase, cystatin and interleukin-3 by B.subtilis 2.
BioBrick standard was applied to the SacB-EAK16-II Fusion Protein.
EAK16-II is a segment from zuotin, a yeast protein. SacB was identified from the initial part of certain preprotein genes that utilises the the Sec-SRP secretory pathway 3. Both components were synthesised as a fusin protein by GeneArt.
Optimized for both B.subtilis and E.coli
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (His-tag)
Untagged version of gfp from Repressilator reporter. See the [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0040 untagged version] for source information.
Added Freiburg-restriction-sites for in frame assembly.
[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]
PlepA is the promoter of the PlepA gene of Bacillus subtilis. It is a constitutive promoter and does not contain a ribosome binding site.
RFP from Discosoma striata (coral) with LAA-ssrA degradation tag (wt)
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012)
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0050, but same tag was also used in paper by Andersen et al., (1998).[2]
Vincent Leonardo, Christopher Wall and Robert Mckiernan
This BioBrick facilitates the switching of Bacillus subtilis cells between rod-shape to L-form, a cell wall-less form of bacteria. It consists of chloramphenicol acetyl-transferase (cat) gene and a xylose-controlled promoter (PxylR) which are flanked by ~300bp pbpB gene at the 5???end and ~300bp of murE gene at 3???end. These flanking sequences allows the integration of the BioBrick into the chromosome by homologous recombination replacing the region containing the end of pbpb gene and the beginning of the murE gene, which are involved in peptidoglycan biosynthesis. This event replaces the constituitive murE promoter with a PxylR upstream of the murE gene. Therefore, when xylose is present cell wall biosynthesis is switched on, giving rod cells. When xylose is not present, the cells will become L-forms. Simply transform the BioBrick into competent B. subtilis cells and through recombination it will integrate into cells chromosome. Plate out the transformants onto 1.5% lysogeny broth agar containing 5??g/ml Chloramphenicol and 0.8 %( w/v) xylose. This will give you rod cells containing biobrick. To get L-forms, inoculate some colonies into a solution containing an equal amount of nutrient broth and MSM. Also inoculate some transformants into the same media with the addition of 0.8 %( w/v) xylose to clarify that the switch BioBrick is working. Use microscopy to check the morphology of the cells in each media. L-forms will appear near spherical and can vary in size considerably.
There were 3 restriction sites that did not meet the RFC10 requirements, two EcoRI sites and one PstI. The first EcoRI restriction site was located 218bp into the whole BioBrick (inside pbpb region), we changed the sequence from GAATTC to GCATTC. The second restriction site was located 2287bp into the BioBrick in the spacer region between PxylR and murE, we changed it from GAATTC to GTATTC. The PstI site was located 197bp into the BioBrick in the pbpb region and was change from CTGCAG to CTTCAG.
The part is the courtesy of Prof. Jeff Errington research group, and all the sequences come from the Bacillus subtilis genomic sequence. The pbpb section in the BioBrick was taken from end of the pbpb gene 1841-2151bp (309bp). Sequence acquired through microbes online VIMSS38051. The murE section was taken from the start of the murE gene 1-331bp. Sequence from microbes online VIMSS38053. The cat gene and PxylR gene were both come from the B. subtilis genome, but can also be found in the BioBrick Registry (BBa_K823020 and BBa_K143014).
Reflectin 1A (LipA) and LALF (LipA) protein for B.subtilis
N-acetylatedGlucosamine based chemotaxis for Bacillus subtilis
RFP from Discosoma striata (coral) with LVA-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LVA-ssrA degradation tag. The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. This tag is commonly attached to repressor proteins for use in various gene networks (e.g., oscillators). The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; protein stability; Km of binding to the protease; temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction sites. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from paper by Andersen et al., (1998).[2]
GFP gene under expresson of the Pveg promoter and spoVG RBS of ''B. subtilis'', with a chloramphenicol resistance marker for ease of selection. Also contains the 5' and 3' amyE integration sequences to allow integration into ''B. subtilis'' at the amyE locus. This device was used by the Imperial iGEM 2008 team to characterise the Pveg promoter and spoVG RBS (Combined part BBa_K143053 ) as part of the Biofabricator ''subtilis'' project.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase. The double terminator, GFP gene and chlorapmphenicol acetyltransferase gene were taken from the registry. The sequence of promoter Pveg and RBS spoVG were taken from papers.
Surfactin generator for B. subtilis
In B. subtilis strains this part can induce the synthesis of surfactin, a lipopeptide with an antimicrobial activity and a powerful surfactant.
This part contains an Bacillus xylose inducible promoter, a RBS for Bacillus and the sfp gene. It can be used to induce surfactin production in Bacillus subtilis strains. However, the sfp gene does not code for the surfactin; it codes for an enzyme which catalyzes the synthesis of the lipoprotein surfactin. In order to overinduce the sfp production and therefore the surfactin synthesis in B. subtilis strains the culture media should be supplemented with xylose.
The sfp gene was isolated from many B. subtilis strains (for example ATCC 21332).
Sufactin generator and biofilm repressor for B. subtilis
Ioana Sandu, Gael Chambonnier, Alex Mizgier, Marion Traouan
This part can be used to induce surfactin production (an antimicrobial lipopeptide) and to repress the biofilm formation in B. subtilis strains.
The part has 3 genes (sfp, abrB and lacI) under the control of the xylose inducible Pxyl promoter.
The part was constructed from the assembly of 3 biobricks: BBa_K802006,BBa_K802007 and BBa_K802008.
25aa with T1 terminator and Constitutive promoter veg
Lf (loctoferrin) is located in many key body fluids , e.g., milk, tears, saliva and other secretions, and in white blood cells ,it has become clear that it is an important part of mammalian defence mechanisms, with a wide range of protective functions.25 amino acid residues is from Lf but has a strong bactericidal, bacteriostatic ability, and the antimicrobial activity than natural LF.We add T1 terminator and Constitutive promoter veg on the 25aa to make it can work regularly.
25aa doesn't contain initiator codon and terminator,so we add a Constitutive promoter veg and T1 terminator .
25aa comes from Lf,which was hydrolysed by protease. We synthesis it by Wu Han Bio-broad Co.Ltd
pBs4S: Bacillus subtilis vector, thrC integration, spec resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. This backbone is a BioBricked version of the B. subtilis vector pDG1731.
This vector is compatible with pBS1C and pBS2E in one strain.
This BioBrick is part of the LMU 2012 igem project BacillusBioBrickBox For handling of B. subtilis vectors, please see here. The transformation into B. subtilis is explained here.
GFP generator. Inducible using IPTG/Lactose.
AmyE integratable PoPS generator (P43-spoVG) (with CmR)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to a chloramphenicol resistance generator in closed transcriptional unit (Parts BBa_K143051 and BBa_K143064 ) and the PoPS generator P43-spoVG ( BBa_K143051 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The chloramphenicol adenyltransferase give resistance to chloramphenicol while the terminator prevents readthrough. The P43-spoVG promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of P43-spoVG was obtained from papers. Chloramphenicol adenyltransferase and the double terminator were obtained from the registry.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the P43-spoVG that was synthesised by GeneArt and Chloramphenicol adenyltransferase and the double terminator that were obtained from the registry.
Pveg is a strong, constitutive promoter of Bacillus subtilis
5??? Integration Sequence for the epsE locus of B. subtilis
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome.The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143006) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The EpsE (aka YveO) locus has to our knowledge never been used for integration into B.subtilis before, but is useful in that it knocks out the potential molecular clutch EpsE gene [1]. In particular, both the 5' and 3' integration sequences for the EpsE locus conatin in-frame stop codons to prevent translation of the gene (if nothing is integrated into the locus, integration also prevents correct EpsE expression). The 5' and 3' integration sequences for the EpsE locus were used to integrate over the EpsE gene and prevent its expression in the Imperial 2008 iGEM project B.sutbilis host. References 1. Blair KM, Turner L, Winkelman JT, Berg HC, and Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 2008 Jun 20; 320(5883) 1636-8. doi:10.1126/science.1157877 pmid:18566286
The EpsE integration sequences were designed from the EpsE (aka YveO) gene's Genbank entry[2] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu DNA polymerase
The 5??? integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosme from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. It was produced by PCR cloning with Pfu DNA polymerase
The plasmid is designed to integrate a cloned insert into the B. subtilis 168 chromosome at the sacA locus. The user inserts the fragment inside the MCS whi
==Part Description== This is a composite part made from Green Fluorescent Protein (GFP) and a double terminator. Green Fluorescent protein (originally from part [http://partsregistry.org/Part:BBa_E0040 BBa_E0040]) that will glow green when excited by the correct wavelengths and thus can be used as a reporter gene. This part is directly fused to a terminator (originally from part [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]) so it is ideal to be fused at the end of your construct. ==Table of Statistics== {| style="color:white; background-color:#0066CC;" cellpadding="20" cellspacing="0" border="2" !Chassis !Monomer/Multimer !Aminoacid Length/Weight !Localisation !Structure !Excitation wavelengths !Emmision wavelengths |- |Prokaryotic and Eukaryotic |Bipartite |274 aminoacids | |beta-barrel(for GFP) hairpins for Terminator |501nm |511nm |} ==References== #Valdivia pmid=8707053
No additional design considerations. The parts were fused together following RFC10 assembly.
GFP originates from jellyfish Aequeora victoria. For more details visit the [http://www.expasy.ch/uniprot/P42212 SwissProt entry]. The double terminator was designed by Reshma Shetty. For more details visis the [http://partsregistry.org/Part:BBa_B0014 original part page].
Promoter ctc is a sigma factor B dependent promoter found in ''B.subtilis''. In ''B.subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. *The context with which we used the promoter ctc, was to take blue light as an input and give '''Polymerase Per Second'''(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in ''B.subtilis'' 3. ===Reference=== #1 pmid=16267279 #2 pmid=3100810 #3 pmid=17575448
Biobrick standard was applied to the promoter ctc sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 23. This sequence was then synthesised by Geneart.
This part is the coding sequence for the spaK gene from Bacillus subtilis ATCC6633. SpaK is the sensor kinase for the subtilin-responsive two component system encoded by the the spaRK operon. SpaK is phosphorylated in response to the presence of subtilin.
CWB domain of B. subtilis major autolysin LytC
Cell wall binding domain and signal peptide (amino acids 1 to 333) of ''Bacillus subtilis'' major autolysin LytC (Uniprot [http://www.uniprot.org/uniprot/Q02114 Q02114]). This domain has been used for displaying heterologous proteins on the cell surface of ''B. subtilis'' (Tsuchiya ''et. al.'', 1999). Used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides by C-terminal fusions to mediate adhesion of ''B. subtlis'' to pathogens.
Due to the N-terminal signal peptide, this part allows only C-terminal fusions of proteins to be displayed on the cell surface. The CWB domain of LytC consists of three N‐terminal type II cell wall‐binding motifs, ranging from position 30 to 318. To minimize possible effects of a translational fusion to the CWB domain, the part includes the downstream unstructered region until position 333. An additional linker can be inserted to allow more structural flexibilty.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytC_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC TTGCGTTCTTATATAAAAGTCCTAACAATGTG LytC_333_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TGAATCTTGATCACCGTGACCC
Pveg + N-acetylatedglucosamine based chemotaxis for Bacillus subtilis
Promoter gsiB is a sigma factor B dependent promoter found in ''B.subtilis''. In ''B.subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter gsiB has been used previously as a read out for the activation of sigma factor B 2. The context with which we used the promoter gsiB, was to take blue light as an input and give '''Polymerase Per Second'''(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in ''B.subtilis'' 3.
Biobrick standard was applied to the promoter gsiB sequence.
GFP (mut3b) with LVA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LVA-ssrA degradation tag. The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part E1010, the tag sequence was obtained from paper by Andersen et al., (1998).[2]
RBS Start-codon SpiderSilkSubunitN1 Strep-tag
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN1) with a Bacillus Subtilis ribosome binding site (RBS) and the Strep-tag infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. The Strep-tag, fused at the N terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin. SubunitN1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites.
reflectin for b.subtilis with LipA signal peptide
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. LipA is a signal peptide from the B. subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[3]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis. Reflectin is a self-assembling protein that has the ability to reflect the sun light which depends on the thickness of the protein layer. Therefore, we thought that this ability could be used as a novel reporter for B.subtilis and E.coli.
Promoter hyper-spank is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter hyper-spank can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The context with which we used the promoter hyper-spank, was to take an input of IPTG and give '''Polymerase Per Second'''(PoPS) as an output. IPTG does not induce the promoter hyper-spank directly, but requires the transcriptional regulator '''LacI''', ( BBa_K413035 ). This means that LacI must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank.
Biobrick standard was applied to the promoter hyper-spank sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 123. This sequence was then synthesised by Geneart.
Anti MazF is an antisense RNA producing sequence which is complemantary to MazF (K302033). MazF is a non-specific ribonuclease coding element which kills Bacillus subtilis. Anti MazF element produces an mRNA which is reverse complemantary to mRNA produced by MazF. Thus, Anti Mazf mRNA binds to MazF mRNA and inhibit its activity for coding MazF protein. With pHyperspank promoter, the production of Anti MazF will be depend on IPTG presence in the medium. If there is IPTG in medium, it will inactivate LacI which inhibit pHyperspank promoter. Thus, Anti MazF will be produced. On the other hand, if there is no IPTG in medium, LacI will inhibit pHyperspank and Anti MazF mRNA cannot be produced. As a result MazF protein will be produced, and kills the bacteria.
Anti MazF is designed as a complemantary sequence to the last 4 bases of the RBS sequence, 6 bases to the intermediate region and to the first 40 bases of MazF coding sequence, tottally 50 bases.
It is synthesized as a reverse complementary sequence to MazF (K302033)
PcotV: B. subtilis promoter regulates spore crust protein cotV
PcotV: B. subtilis promoter regulates spore crust proteins and is activated in late sporulation. PcotYZ regulates the crust protein cotZ and cotY. We used this promoter to control the fusion proteins for display on B. subtilis spores. We experienced weaker strength as the related promoter PcotYZ [http://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins Part:BBa_K823030] see all the details about the LMU-Munich 2012 project here: http://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins
We used this part for the regulation of spore crust fusionproteins in B. subtilis
IPTG-inducible arginase production for ''Bacillus subtilis''.
Two EcoR1 restriction sites were removed from the ''rocF'' coding sequence.
The ''rocF'' sequence was taken from ''Bacillus subtilis'' 168 genomic DNA, by PCR. Pspac oid,. .... assembled by the Gibson method...
PcomC, a CSP inducible promoter derived from Streptococcus pneumoniae
cotZ: B. subtilis spore crust protein
This part codes for one of the outer most spore crust proteins in B. subtilis. We designed this part in the Freiburg standard (Assembly 25) to use it as a platform for protein display on B. subtilis spores.
This part is compatible with the freiburg standard (Assembly 25) and can be used for fusion proteins. We used the promoter PcotYZ to regulate transcription.
This promoter can be induced by IPTG. Wild type LacI binding site is replaced with a perfect palindromic "oid" operator sequence which increases the repression by LacI. This biobrick is an enhancement of Biobrick BBa_K090501 which has the sequence from pmutin2.
pmutin2's genbank accession number is AF072806. Although there is no sequence information for pmutin4, it can be derived from pmutin2's sequence information by changing the O1 lacI binding site with the "oid" sequence.
This sequence is taken from pmutin4 integration vector. pmutin2's lacI binding site was changed with the "oid" sequence to create pmutin4.
Signal peptide of B. subtilis major autolysin LytC
Signal peptide (amino acids 1 to 25) of ''Bacillus subtilis'' major autolysin LytC (N-acetylmuramoyl-L-alanine amidase, Uniprot [http://www.uniprot.org/uniprot/Q02114 Q02114]). Targets proteins for secretion via the Sec pathway (Tjalsma ''et. al.'', 2000).
This part was generated by amplification from BioBrick [http://parts.igem.org/Part:BBa_K1351006 BBa_K1351006] with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytC_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC TTGCGTTCTTATATAAAAGTCCTAACAATGTG LytcSP_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT ATCGGCCAAAGCTGTTGGC
pSBBs4S: Empty backbone for integration into Bacillus subtilis thrC locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1731.
This plasmid can be used as empty backbone vectors like pSB1C3 but is able to integrate in B.subtilis. The clonging site also includes a rfp for selection.
Ciprofloxacin resistance cassette in integration plasmid (BBa_K823023)
This part is a ciprofloxacin resistance cassette. It is containing a PAtpI constitutive promoter, ciprofloxacin resistance gene qnrS1 and double terminator. The promoter PAtpI ([http://parts.igem.org/Part:BBa_K1930005 ''BBa_K1930005'']) is a constitutive promoter which has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 minutes of the germination of B. subtilis. atpA gene is part of an operon (atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC), therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. Ciprofloxacin resistance gene qnrS1 is found naturally in E. coli, and other Gram-negative strains. qnr genes code for pentapeptide repeat proteins.??These proteins reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase enzyme from the inhibitory effect of quinolones. Double terminator ([http://parts.igem.org/Part:BBa_B0015 ''BBa_B0015'']) is the most commonly used terminator. For more information see here: http://parts.igem.org/Part:BBa_B0015.
Ciprofloxacin resistance cassette: Ordered as gBlock. IDT (Integrated DNA technologies). BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023.
AmyE integratable PoPS generator (P43-gsiB) (with CmR)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to a chloramphenicol resistance generator in closed transcriptional unit (Parts BBa_K143050 and BBa_K143064 ) and the PoPS generator P43-gsiB ( BBa_K143050 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The chloramphenicol adenyltransferase give resistance to chloramphenicol while the terminator prevents readthrough. The P43-gsiB promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of P43-gsiB was obtained from papers. Chloramphenicol adenyltransferase and the double terminator were obtained from the registry.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the P43-gsiB that was synthesised by GeneArt and Chloramphenicol adenyltransferase and the double terminator that were obtained from the registry.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral) with barcode
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
This part is essentially identical to BBa_E1010 , but has a point mutation in the barcode. Barcodes are discontinued, but composite parts using this part will have the mutated barcode.
Promoter gsiB and RBS gsiB for B. subtilis
Sigma B dependant promoter gsiB( BBa_K143011 ) coupled to the strong Ribosome Binding Site gsiB( BBa_K143020 ) from ''B. subtilis''. In ''B. subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. PgsiB has been used to take an input of blue light and give a '''Ribosomes per second'''(RiPS) output. To work as a blue light receiver correctly, over-expression of the blue light receptor '''YtvA''' ( BBa_K143037 ) is required. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter gsiB was obtained from the ''B. subtilis'' genome and published papers1 2. RBS-gsiB were obtained from papers3 and the sequence synthesised by GeneArt
RBS Start-codon SpiderSilkSubunitE1 SpiderSilkSubunitE2 SilkTail Strep-tag stop-codon
The ferric uptake repressor of Bacillus subtilis called YqkL is a regulator that when in contact with iron will bind and repress a regulatory sequence (the Fur box).
We sequenced the Fur box. This can be added in front of genes to provide regulation when in contact with iron.
38aa and 25aa fusion peptide with T1 terminator and Constitutive promoter veg
Human lysozyme (hLZ), the actual protein in breast milk,exhibits microbicidal activity to various degrees against several bacterial strains.The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria.Evidence shows that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. Lf (loctoferrin) is located in many key body fluids , e.g., milk, tears, saliva and other secretions, and in white blood cells ,it has become clear that it is an important part of mammalian defence mechanisms, with a wide range of protective functions.25 amino acid residues is from Lf but has a strong bactericidal, bacteriostatic ability, and the antimicrobial activity than natural LF.
Codon optimization has been done,so the protein will be expressed normally in Ecoli.
25aa comes from Lf,which was hydrolysed by protease.38aa comes from Human Lysozyme. They were both synthesized in Wuhan Bio-Broad Co.Ltd
pBS2E: Bacillus subtilis vector, lacA integration, MLS resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli.
Compatible with pBs1C and pBs4S in one strain.
This backbone is a BioBricked version of the B. subtilis vector pAX01. This BioBrick is part of the LMU 2012 igem project [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick'''B'''ox] Reference: [http://www.ncbi.nlm.nih.gov/pubmed/11274134 H??rtl ''et al.'']
Promoter Xylose is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give '''Polymerase Per Second'''(PoPS) as an output. Promoter xylose is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give '''Polymerase Per Second'''(PoPS) as an output. Xylose does not induce the promoter xylose directly, but requires the transcriptional regulator '''XylR''', ( BBa_K143036 ) This means that XylR must be constitutively expressed in ''B.subtilis'' in order to use the promoter xylose.
Biobrick standard was applied to the promoter xylose sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 12. This sequence was then synthesised by Geneart.
(Chassis B. subtilis, carrier plasmid pSBBS0K) Length: 6843 bp Background: pNagP is the promoter of the NagP gene, which expresses a transporter of N-acetylglucosamine-6-phosphate in Bacillus subtilis (http://www.biocyc.org/gene?orgid=BSUB&id=BSU07700). This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
The BBa_K1937006 part was designed to demonstrate the induction of pNagP by NAG. It belongs tothe antifungal module in the Paleolitis project of iGEM Toulouse 2016 (http://2016.igem.org/Team:Toulouse_France). The part include the pNagP promoter and its RBS (position 840,455 to 840,656 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping. This part is a subcloning of BBa_K1937005 pNagP part in pSBBS0K_mini plasmid (BBa_K1937001).
In bacillus subtilis, this promoter has been reported to induce expression in presence of N-acetylglucosamine 6-phosphate (Bertram et al., 2011: https://www.ncbi.nlm.nih.gov/pubmed/21602348). This NAG molecule is the major component of chitin, a characteristic molecule of fungi.
Biofilm repressor for B. subtilis strains
This part could be used to impede biofilm formation in B. subtilis.
The part has a Bacillus RBS before the abrB CDS.
The abrB gene was isolated from Bacillus subtilis.
This is the weak version of the consensus RBS for Bacillus subtilis. The sequence is present in front of some B. subtilis operons, but is expected to give weaker expression than the consensus sequence. The consensus sequence AAAGGAGG (BBa_K090505) was changed to AGAGGTGG for this part.
The part must be 8 nucleotides away from the first methionine of the coding sequence, and so we added 'TGT' downstream of the actual binding sequence: AGAGGTGG. This, combined with the truncated non-CDS BioBrick prefix scar, gives exactly 8 'spacer bases' between the 3' end of the RBS and the start of the coding sequence.
Promoter 43 a constitutive promoter for B. subtilis
Promoter 43 is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. This promoter has been shown to be recognized and active during the exponential and lag phases of growth. It has been hypothesized that the ability to recognize the promoter in exponential and lag phase of growth is due to the recognition of the promoter by both sigma factor 55 (the major sigma factor) and sigma factor 37 (the lag phase sigma factor) 1. The P43 promoter has been previously used for constitutive expression of exogenous genes within ''B.subtilis'' vectors 2. The context with which we used the promoter P43 is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include the binding sites for both the sigma factor A and B. In addition the biobrick standard was applied to the promoter P43 sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 2. This sequence was then synthesised by Geneart.
Promoter ctc and RBS spoVG for B. subtilis
Sigma B dependant promoter ctc( BBa_K143010 ) coupled to the strong Ribosome Binding Site spoVG( BBa_K143021 ) from ''B. subtilis''. In ''B. subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. Pctc has been used to take an input of blue light and give a '''Ribosomes per second'''(RiPS) output. To work as a blue light receiver correctly, over-expression of the blue light receptor '''YtvA''' ( BBa_K143037 ) is required. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter ctc was obtained from the ''B. subtilis'' genome and published papers1 2. RBS-spoVG were obtained from papers3 and the sequence synthesised by GeneArt
Ferric uptake repressor box (Fur box). When the ferric uptake repressor binds to it, it represses transcription.
Fur boxes differ, we used the consensus sequence which per se is not an actual sequence occuring within B. subtilis but rather the strongest sequence that Fur protein would bind to.
Bacillus subtilis Fur box consensus sequence.
Anti MazF construct (pHyperspank + Anti MazF + Double Terminator)
PlepA is a constitutive promotor in Bacillus subtilis (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.
The sequence has been verified by sequencing
http://parts.igem.org/Part:BBa_K823002 http://parts.igem.org/Part:BBa_K143021
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitE1) with Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitE1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites. The amino acid sequence is based on the adapted MaSp2 sequences discribed in the paper of Brooks et. al. 2008.
For N-terminal fusion proteins with cgeA (display on B. subtilis spores).=='''Sporo'''vector== Our '''Sporo'''vector is specially designed to give you the opportunity to easily create Sporobeads with any proteins you can think of. It is available as BioBrick in pSB1C3 and can be cloned in any of our empty ''B. subtilis'' vectors. We also offer it ???precloned??? in pSBBs4S which in this version lacks ''NgoM''IV restriction sites. The '''Sporo'''vector was created by ligation of three cut PCR-products. It is designed to allow N-terminal fusion of genes in Freiburg standard ([http://dspace.mit.edu/bitstream/handle/1721.1/45140/BBF_RFC%2025.pdf?sequence=1 RCF25]). Therefore, the gene to be inserted must be cut with ''Xba''I and ''Age''I and the vector must be digested with ''Xba''I and ''NgoM''IV. By this digest, the RFP cassette will be removed from this vector to allow easy selection for the integration of the new insert. After ligation, there is a scar of six nucleotides between both genes but no restriction site left. The gene fusion is controlled by the P''cot''YZ-promoter which was the strongest one in our promoter evaluations. The C-terminal gene part is ''cge''A, one of the two known spore crust proteins. The vector is cloned but has not been tested with an insert, yet. We are working on further '''Sporo'''vectors to offer also fusion with ''cot''Z as well as C-terminal fusions.
For N-terminal fusion proteins with cgeA (display on B. subtilis spores)
Encodes a stable non-specific ribonuclease in ''Bacillus Subtilis''. It is used in conjungtion with ''mazE'' in ''Bacillus Subtilis'' to provide a toxin-antitoxin in various stressful conditions. When transcribtion of both genes is turned off (as both are under contol of same promoter) ''mazE'' will be degraded faster than ''mazF''. There is then no inhibiton of ''mazF'', killing the cell.
Sequence isolated based upon the work of Pellegrini, O., Mathy, N., Gogos, A., Shapiro, L., and Condon, C. (2005) The ''Bacillus subtilis'' ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. ''Mol Microbiol'' 56: 1139???1148.
pSBBs3C-luxABCDE (lux reporter vector for B.subtilis)
This part is a reporter vector for Bacillus subtilis. It integrates at the sacA locus of B.subtilis genome. It has a ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for selection in B. subtilis. The multiple cloning site has a rfp for selection and is followed by the luxABCDE gene cluster for luminescence reporter assays. This BioBrick is part of the LMU-Munich 2012 igem project Bacillus BioBrickBOX http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This vector is an igem compatible version of the pAH328 Bacillus reporter vecotr.
The BBa_K1937003 part was designed to demonstrate the induction of pNagA by NAG. It belongs tothe antifungal module in the Paleolitis project of iGEM Toulouse 2016 (http://2016.igem.org/Team:Toulouse_France). pNagA is the promoter of the NagA gene, which expresses a N-acetylglucosamine-6-phosphate deacetylasein in Bacillus subtilis In bacillus subtilis, this promoter has been reported to induce expression in presence of N-acetylglucosamine 6 phosphate. This NAG molecule is the major component of chitin, a characteristic molecule of fungi.
The part include the pNagA promoter and its RBS (position 3,595,156 to 3,595,356 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping.
D'o?? vient le promoteur, le RBS, la s??quence codante... la s??quence en tant que telle aussi
This part codes for the chloramphenicol acetyltransferase (cat) gene used in Bacillus subtilis for resistance against the antibiotic chloramphenicol. We constructed it in the freiburg standard (Assembly 25) for in-frame fusion-proteins. There is a Shine-Dalgarno Sequence (RBS) optimized for Bacillus subtilis included between the XbaI and NgoIV restriction site. You can use it both N-and C terminal fusions.
We constructed the part in the freiburg standard (Assembly 25). We included a Bacillus optimized RBS between the XbaI and NgoIV restriction site.
Plasmid: Bacillus subtilis reporter vector pAC6
PcotYZ - cotZ (-2aa) - gfp - Terminator
This composite part expresses a gfp fusion protein on a B. subtilis spore.
This part is cloned in the B. subtilis pSBBs1C backbone vector for direct integration in the amyE locus of B. subtilis.
genomic B. subtilis, registry (Termiantor), Plasmid (pGFPamy)
RFP from Discosoma striata (coral) with AAV-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part E1010) with added AAV-ssrA degradation tag (part I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: 1) ClpXP and ClpAP protease and SspB mediator concentrations; 2) Protein stability; 3) Km of binding to the protease; 4) Temperature [3]. [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3]Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part E1010, tag sequence was obtained from part I11012, but same tag was also usead in paper by Andersen et al., (1998).[2]
AmyE integratable PoPS generator (Pveg-gsiB)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to the PoPS generator Pveg-gsiB ( BBa_K143052 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The Pveg-gsiB promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of Pveg-gsiB was obtained from papers.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the Pveg-gsiB that was synthesised by GeneArt
This BioBrick contains the coding sequence for silk tail with a Strep-tag behind it and ends with a stop codon. The Tail ??????. The Strep-tag, fused at the C terminal, is used both as a binding tag to use to inidicate its production and as a binding component to biotin.
''Bacillus subtilis'' sortase YhcS (UniProt [http://www.uniprot.org/uniprot/P54603 P54603]), responsible for anchoring its substrae YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]) covalently to the peptidoglycan wall of ''B. subtilis''. A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the ''B. subtilis'' cell wall (Liew ''et. al.'', 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of ''B. subtlis'' to pathogens. It consists of four components: the sortase substrate with a signal peptide (this part), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain ([http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 BBa_K1351011]), whose overexpression increases the efficiency of the surface display. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. YhcS_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC GTGAAAAAAGTTATTCCACTATTCATCATTGC YhcS_SNP_rev: gatcCTGCAGcggccgctACTAGTa TTAAGTCACTCGTTTTCCATATATAATATAGCG An EcoRI site was removed by changing T to C at position 399 without altering the protein sequence by fusion PCR with the following mutagenesis primers: YhcS_EcoRI435mut_fwd: CGTCTTCTCCTGTCATATGGAGAATTtACATATGAAATTGTAAAAACAAAAATTGTCG YhcS_EcoRI435mut_rev: CGACAATTTTTGTTTTTACAATTTCATATGTaAATTCTCCATATGACAGGAGAAGACG
AmyE integratable PoPS generator (P43-spoVG)
AmyE 5' Integration sequence( BBa_K143001 ) coupled to the PoPS generator P43-spoVG ( BBa_K143051 ). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence( BBa_K143002 ) is cloned onto the 3' end of the construct. The P43-spoVG promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of P43-spoVG was obtained from papers.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the P43-spoVG that was synthesised by GeneArt.
Promoter 43 and RBS gsiB for B. subtilis
Constitutive promoter 43( BBa_K143013 ) coupled to the strong Ribosome Binding Site gsiB( BBa_K143020 ) from ''B. subtilis''. P43-gsiB can be used in the context of a Polymerase per second (PoPS) output generator.
The sequence of P43 and RBS-gsiB were obtained from papers1 2 and the sequence synthesised by GeneArt
RBS + Antifungal GAFP-1 + Double terminator
RFP gene under expresson of the P43 promoter and spoVG RBS of ''B. subtilis'', with a chloramphenicol resistance marker for ease of selection. Also contains the 5' and 3' amyE integration sequences to allow integration into ''B. subtilis'' at the amyE locus. This device was used by the Imperial iGEM 2008 team to characterise the P43 promoter and spoVG RBS (Combined part BBa_K143051 ) as part of the Biofabricator ''subtilis'' project.
The 5' and 3' amyE integration sequences were PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase. The double terminator, RFP gene and chlorapmphenicol acetyltransferase gene were taken from the registry. The sequence of promoter P43 and RBS spoVG were taken from papers.
beta-Neurexin-derived peptide binding to S. aureus adhesin SdrC
Short peptide of 11 amino acids corresponding to the N-terminus of the human protein neurexin beta-1. It includes the binding site (HHIHHFH) for the Staphylococcus aureus adhesin SdrC. Displayed on the surface of Bacillus subtilis, it shoud enable B. subtilis to bind specifically to S. aureus.
Oligos were designed as precut by EcoRI and PstI to shorten the synthesized fragments and to simplify the cloning into pSB1C3.
The part was generated by annealing of artificially synthesized oligonucletides: Nrx1b_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCGCACCACATTCACCACTTTCACGGATCA ACCGGTtaatACTAGTagcggccgCTGCA Nrx1b_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT TGATCCGTGAAAGTGGTGAATGTGGTGCGC CATGCCGGCttcctccttacTCTAGAagcggccgcG
Membrane bound protein that binds subtilin and prevents it from penetrating the cell membrane to form pores.
Overlaps with ''spaF'' coding sequence at the 3' end
This is an efficient termintor sequence from Bacillus that normally terminates the rrnO-16S-trnO-Ile-trnO-Ala-rrnO-23S-rrnO-5S operon.
This terminator forms part of the complex device BBa_K104001
The sequence was derived from DBTBS http://dbtbs.hgc.jp/COG/prom/rrnO-16S-trnO-Ile-trnO-Ala-rrnO-23S-rrnO-5S.html
Promoter xyl and RBS spoVG for B subtilis
Inducible promoter xyl( BBa_K143014 ) coupled to the strong Ribosome Binding Site spoVG( BBa_K143021 ) from ''B. subtilis''. Pxyl-spoVG can be used to take an input of xylose and give a '''Ribosomes per second''' (RiPS) output generator. Xylose does not directly induce the expression of the promoter xyl, but requires the transcriptional regulator '''XylR''', ( BBa_K143036 ). This means that XylR must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank as an inducible promoter. XylR is naturally expressed by ''B. subtilis'' but should be upregulated to increase efficiency. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter xyl and RBS-spoVG were obtained from papers1 2 3 and the sequence synthesised by GeneArt
This BioBrick contains the coding sequence for part of the spider silk protein (SubunitN1) with Bacillus Subtilis ribosome binding site (RBS) infront of it. The RBS is there to allow the ribosomes to bind and start to translate the DNA. SubunitN1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtillus 168. The primary goal of the algorithm was to optimize the codon selection based on their availbility scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tetrary goal was to minimize the number of restriction sites.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Campbell et al., PNAS v99 p7877 URL
Arabinose controlled protein degradation device
The purpose of this system is to control the expression of sspB, the degradation controller. The system is inducible by arabinose, therefore we can alter the arabinose levels to regulate the degradation of proteins with sspB degradation tag
sspB Biobrick is inserted after araR promoter+rbs system
LALF for Bacillus subtilis with LipA signal peptide
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. LipA is a signal peptide from the B. subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[3]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis. Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. The wedge- shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and it has been proposed that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.
B.subtilis and E.coli Episomal Shuttle Vector with Consitutive RFP to express in E.coli
This episomal vector has within its BioBrick region a constitutively expressed RFP gene in E.coli. It uses LacI promoter, B0034, mRFP1 (E0010) and B0015.
SinR protein is the master regulator of biofilm formation in Bacillus subtillis. In the active state, it represses biofilms.
We used an E. coli vector to accept the sequence. Designing a BioBrick compatible Bacillus subtilis vector took a lot of effort.
RBS+GFP for expression in Bacillus subtilis
It is succesfully joined B.subtilis RBS with GFP reporter protein. Can be used to measure strenght of B.subtilis promoters. In order to work with it you must first add appropriate terminator at the end. We recommend BBa_K780000 - a terminator designed for use with B.subtilis.
RBS was taken from 'host-takover module' from B.subtilis Bacteriophage SPO1 and GPF is derived from jellyfish Aequeora victoria (BBa_E0040).
spaS Subtilin (antimicrobial peptide, Freiburg standard)
Subtilin Lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. The bactericidal activity of lantibiotics is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores.
LipA-Human Elastin (EP20-24-24) Fusion Protein
Elastin is a polymeric extracellular matrix protein found in tissues that require the ability to extend and recoil. Examples of elastin containing tissues include arteries, lungs, ligaments and skin. Construct EP20-24-24 for human elastin polypeptide consists of distinct exons which code for alternating hydrophobic regions and crosslinking domains from the human elastin polypeptide gene 1. Under appropriate conditions of temperature and ionic strength, elastin polypeptide undergoes a self-aggregation process known as coacervation. Coacervation is usually induced by an increase in temperature and causes the protein to separate from the solution as a second phase. Unlike most proteins which undergo denaturation when the temperature of the solution increases, elastin polypeptides become more ordered through coacervation 2. LipA is a signal peptide from the B.subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway3. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by ''B. subtilis''.
BioBrick standard was applied to the LipA-Human Elastin(EP20-24-24) Fusion Protein.
All exons in EP20-24-24 are derived from the human elastin polypeptide gene. EP20-24-24 was used to study the effect of various combinations of exons on coacervation of elastin polypeptide. LipA originated from the B. subtilis genome4. Both components were produced as a fusion protein by GeneArt.
This is the ribsome binding site for the spaR gene. spaR encodes the response regulator for the two component system encoded by the spaRK operon that senses the quorum peptide subtilin.
GFP (mut3b) with DAS-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added DAS-ssrA degradation tag (see part BBa_M0052). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag encodes peptide sequence AANDENYADAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0052.
Anti MazF - Antisense RNA to inhibit MazF activity in B.subtilis
Anti MazF is an antisense RNA producing sequence which is complemantary to MazF (K302033). MazF is a non-specific ribonuclease coding element which kills Bacillus subtilis. Anti MazF element produces an mRNA which is reverse complemantary to mRNA produced by MazF. Thus, Anti Mazf mRNA binds to MazF mRNA and inhibit its activity for coding MazF protein.
This part is designed as a complemantary sequence to the last 4 bases of the RBS sequence, 6 bases to the intermediate region and to the first 40 bases of MazF coding sequence, tottally 50 bases.
Anti MazF is an antisense RNA producing sequence which is complemantary to MazF (K302033). MazF is a non-specific ribonuclease coding element which kills Bacillus subtilis. Anti MazF element produces an mRNA which is reverse complemantary to mRNA produced by MazF. Thus, Anti Mazf mRNA binds to MazF mRNA and inhibit its activity for coding MazF protein. This part can be used with any promoter according to regulation mechanism.
pBS1C: Bacillus subtilis vector, amyE integration, CAM-resistance
This vector integrates into the amyE-site of Bacillus subtilis via double recombination. The two recombination sites flank a standard BioBrick multiple cloning site (with RFP-cassette), as well as a chloramphenicol resistance cassette (5 ??g/ml). The backbone does not contain the restriction sites EcoRI, XbaI, SpeI, PstI, NgoMIV nor AgeI. The vector is replicative in Escherichia coli and provides resistance to ampicillin (100 ??g/ml). Before transformation into B. subtilis, this vector should be linearized with ScaI.
This is one of three empty vectors with compatible integration loci and resistance cassettes to allow combination of up to three constructs of choice in one single strain.
Promoter hyper-spank and RBS spoVG for B. subtilis
Inducible promoter hyper-spank( BBa_K143015 ) coupled to the strong Ribosome Binding Site spoVG( BBa_K143021 ) from ''B. subtilis''. Phyperspank-spoVG can be used to take an input of IPTG and give a '''Ribosomes per second''' (RiPS) output generator. IPTG does not directly induce the expression of the promoter hyper-spank, but requires the transcriptional regulator '''LacI''', ( BBa_K143033 ). This means that LacI must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank as an inducible promoter. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of promoter hyperspank was obtained from the ''B. subtilis'' integration vector pDR111 and RBS-spoVG were obtained from papers1 and the sequence synthesised by GeneArt
Phyperspank-spoVG was synthesised by GeneArt