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).
constitutive expression of anti-Norovirus GII.4 scFv fused to INPNC with 6xHis tag
anti-Norovirus GII.4 scFv fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a single chained variable fragment(scFv) of a norovirus GII.4 strain fused to surface expression anchoring domain(INPNC), connected by a poly-histidine tag to be easily identified by Western blotting. For more information, please visit our Wiki page.
Silent mutation was done to remove PstI site.
Promoter and RBS The INPNC surface expression component derives from Pseudomonas Syringae. We used parts BBa_K811005 from iGEM12_Penn. scFv is made from fusing variable region (VH and VL chain) of a human anti-norovirus antibody with a linker peptide. We have been given the scFv-encoding plasmid (NoV GII.4 strain-specific antibody 12A2 (vector: pSCCA5-E8d)) from Dr. Kyoko Moriguchi of the Fujita Health University Department of Virology Parasitology.
LsrA of LuxS/AI-2 signaling pathway in Salmonalla
In a process called quorum sensing, bacteria communicate with one another using secreted chemical signaling molecules termed autoinducers. As the density of the bacterial population increases, so does the concentration of secreted autoinducer molecules. When the concentration of the autoinducer reaches a critical threshold, it diffuses back into the cell and activates or represses certain target genes. An autoinducer called AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In the LuxS/AI-2 signaling system of Salmonella enterica serovar Typhimurium, AI-2 response involves genes named lsr (LuxS regulated) that encode ATP binding cassette-type transporter. And LsrA is ATP-binding protein of the ABC transporter. In our project, we set this part under a nisA promoter for the final purpose of constructing an integrated AI-2 response pathway in our engineering bacteria.
The lsrA gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR.We added restriction enzyme sites when designing primers,and the assembly is done through the use of restriction sites (cutting and ligating).
It comes from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2(Gene ID: 1255601)
Outer Membrane Protein X (OmpX) with BamHI-linker and mNeonGreen
This part consists of three subparts, namely the uter membrane protein OmpX, a BamHI linker and the fluorophore mNeonGreen. OmpX is an outer membrane protein with the C- and N-termini in the intracellulair domain. To be able to use OmpX as a scaffold, a non-natural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction with DBCO functionalized groups. The BamHI linker is a 213 bp long flexible GGSGGS linker. Using the restriction enzyme BamHI, the linker can become 45 bp shorter. mNeonGreen is a yellow-green fluorescent protein. For a fluorescene measurement, the mNeonGreen fluorophore can be excitated with a laser with a wavelenght of 480 nm and readed out at 517 nm.
The sequence is redesigned considering codon optimalization. Also restriction sites for biobricking and classical cloning are deleted. From the BamHI linker, the coding sequecne for GGSGGS is redesigned causing that only two BamHI restriction sites remain.
OmpX is a outer membrane protein naturally occuring in wildtype E.coli. The BamHI linker is inspired by the article "Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Liners" by Toon H. Evers et all from 29 August 2006. mNeonGreen naturally occurs in the organism Branchiostoma Lanceolatum.
The motA gene codes for a part of the flagellar motor complex. MotA proteins represent the proton conducting component of this molecular machine. Thus it is necessary for flagellar rotation.
*''motA'' was amplified from genomic DNA of ''E. coli'' DH10B via PCR using the following primers: **Fwd: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGACCGGTGGAAGCCTTGG **Rev: GTTTCTTCCTGCAGCGGCCGCTACTAGTATCATGCTTCCTCGGTTGTCG
* The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B, complete genome (CP000948.1).
This gene codes for MotB, a part of the flagellar motor. MotA and MotB constitute the stator and are required for the flow of ions and torque generation.
* motA was amplified from genomic DNA of E. coli DH10B via PCR using the following primers: ** Fwd: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGAATCAAGCGCATCC ** Rev: GTTTCTTCCTGCAGCGGCCGCTACTAGTATCACCTCGGTTCGGCTGATGG
motA under the control of constitutive promoter J23112
The ''motA'' gene codes for a part of the flagellar motor complex. MotA proteins represent the proton conducting component of this molecular machine. Thus it is necessary for flagellar rotation. Here we used 3 different constitutive [http://partsregistry.org/Part:BBa_J23100 Anderson promoters] from the 2006 Berkeley group to test how different levels of constitutive ''motA'' expression affect the motility or flagellum size of ''E. coli''.
*''motA'' was amplified from genomic DNA of ''E. coli'' DH10B via PCR using the following primers: **Fwd: 5'-gtttcttcgaattcgcggccgcttctagatgaccggtggaagccttgg-3' **Rev: 5'-gtttcttcctgcagcggccgctactagtatcatgcttcctcggttgtcg-3'
* ''motA'' was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). * J23100 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
AraC+Pbad promoter+RBS+signal peptide phoA+Cellulase+Linker+estA membrane protein
The composite part includes a functional expression vector for autotransporter membrane protein estA, with a linker united to a cellulase, attached to a membrane signaling peptide. This was our final construct missing a terminator
mtrCAB - gene cluster from S. oneidensis for cytochrome expression
Minimal set of genes for the expression of working cytochromes. Allows the organism to transfer electrons to its outer membrane.
Two illegal PstI restriction sites, which had to be removed by specific primer extensions.
constitutive expression of CBDcex fused to BclA with 6xHis tag
CBDcex fused to BclA with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBD) fused to surface expression anchoring domain(BclA), connected by a 6xHis tag to be easily identified by Westernblotting. For more information, please visit our Wiki page.
The stop codon for this part is not in the coding region, but in the suffix region.
Promoter and RBS:BBa_K1933004 The BclA surface expression component derives from Bacillus anthracis(34F2), it is a hair-like protein that covers B. anthracis??? spores. We used parts BBa_K1933000 from iGEMKyoto 2016 for construction. We outsourced chemical synthesis of this part for construction. CBDcex derives from Cellulomonas fimi, and is part of an exoglucanase secreted by this organism. We used parts BBa_K1321342 from iGEM Imperial 2014 for construction.
motA under the control of constitutive promoter J23114
* ''motA'' was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). * J23114 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
Outer membrane porin OprF from Pseudomonas fluorescens
By heterologous expression of the pore-forming transmembrane-protein (or porine) OprF from Pseudomona fluorescens a further optimization of the E. coli membrane will be achieved. E. coli possesses several own porines, for example OmpF and OmpC. But these naturally occurring porines are only permeable for molecules smaller than 600 Da, which decreases the range of usable mediators significantly. Opposed to that, porine OprF from P. fluorescence forms one of the biggest known pores in the outer bacterial membrane with a permeability of up to 3000 Da, thus improving the electron shuttle mediated extracellular electron transfer (ETT) and enabling the usage of alternative mediators such as riboflavin (vitamine B2).
EnvZ* is a transmenbrane kinase that phosphorylates ompR the natural inhibitor of pFlhDC independently of any outer signal.
bacterial genome of mutated (I281S) strain for EnvZ.
Sequence for chemokine receptor CD4, including signal peptide.
Outer membrane porin OprF from Pseudomonas fluorescens with medium Anderson promotor and weak RBS
CP47 His-tagged with Lac Promoter synthesized by IDT DNA Inc.
INPNC codes for N- and C- terminal domain of Ice-Nucleation Protein from Pseudomonas syringae, connected by a 6??His tag to be easily identified by Western blotting. [1]This is used as an anchoring motif for the cell surface display of passenger domain. INPNC does not have signal peptide for anchoring on the cell surface[2] Surface displayed passengers retain enzyme activity even when it is linked to INPNC with 6?? His tag. For more information, please visit our Wiki page.
AgrC protein coding sequence. AgrC is main component of S.aureus agr quorum sensing system.
AgrC is a transmembrane protein, which is the sensor molecule of a typical two-component signal system in S.aureus. AgrC possesses several key amino acid motifs typical of histidine protein kinase sensor. The AgrC sensor kinase can specifically binds to corresponding AIP, which secreted only from specific S.aureus, and the composite of AgrC and AIP then leads to phosphorylation of AgrA. AgrA in its phosphorylated sate activates transcription from both P2 and P3, leading to the production of GFP and 3OC6HSL. Thus we can detect the presence of S.aureus expediently by observing the expression of GFP.
we employed site-specific mutagenesis(AgrC-I A101T) to replace a amino acid residue in AgrC-I so that the mutated AgrC can bound to both AIP-I and AIP-IV.
EstA : inactive fusion protein for bacterial surface expression
Marie Burghard, Henrik Cordes, Jascha Diemer, Adrian Eilingsfeld, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling
This Esterase A is a inactivated membrane bound esterase from Pseudomonas putida. Its active site is mutated so it shows no more hydrolytic activity. It can be used for cell surface display like its shown in the following paper: Taek Ho Yang, Jae Gu Pan, Yeon Soo Seo, Joon Shick Rhee Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli Appl Environ Microbiol. 2004 December; 70(12): 6968???6976. doi: 10.1128/AEM.70.12.6968-6976.2004
This certain variant of EstA should be used for cell surface expression and displaying of almost every kind of enzyme. There is no ATG at its 5' because its an C-terminal fusion protein. If you want to use you need to design it in the following way: N - Peri plasmatic signal sequence - Protein of interest - EstA - C We did not use any kind of BioBrick Assembly due to the generation of stop codons. If you would like to fuse EstA to any kind of protein, please use SOE PCR or an alternative kind of assembly strategy.
Our parts derives from an established inactive EstA mutant on pEST100 vector, which was used in the following paper: Becker, S., Theile, S., Heppeler, N., Michalczyk, A., Wentzel, A., Wilhelm, S., Jaeger, K.-E., et al. (2005). A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS letters, 579(5), 1177???82. doi:10.1016/j.febslet.2004.12.087
Outer membrane porin OprF from Pseudomonas fluorescens with medium Anderson promotor and medium RBS
Inducible High Expression His-Tagged Photosystem II CP47 subunit
This part includes a LacY coding sequence on the backbone J61002
Outer membrane porin OprF from Pseudomonas fluorescens with strong Anderson promoter and strong RBS
constitutive expression of CBDclos fused to BclA with 6xHis tag
CBDclos fused to BclA with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBD) fused to surface expression anchoring domain(BclA), connected by a 6xHis tag to be easily identified by Western blotting. For more information, please visit our Wiki page.
The BclA surface expression component derives from Bacillus anthracis(34F2), it is a hair-like protein that covers B. anthracis??? spores. We used parts BBa_K1933000 from iGEMKyoto 2016 for construction. We outsourced chemical synthesis of this part for construction. CBDclos derives from Clostridium cellulovorans, and is part of a multi-unit cellulosomes. We used parts BBa_K1321341 from iGEM Imperial 2014 for construction.
Outer membrane porin OprF from Pseudomonas fluorescens with PT7 and RBS
E1 is a membrane protein that comes from HCV. The receptors on the liver recognize this protein, which leads to endocytosis of the particle displaying the E1. The protein can be used for specific targeting to the liver.
We had to make sure the amino acid sequence produced is the same as the amino acid sequence of E1 that we found online.
motA under the control of constitutive promoter J23100
motB under the control of constitutive promoter J23100
* ''motB'' was amplified from genomic DNA of ''E. coli'' DH10B via PCR using the following primers: ** Fwd: 5'-gtttcttcgaattcgcggccgcttctagatgaagaatcaagcgcatcc-3' ** Rev: 5'-gtttcttcctgcagcggccgctactagtatcacctcggttcggctgatgg-3'
* The part was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). * J23100 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
constitutive expression of CBDcex fused to INPNC with 6xHis tag
CBDcex fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBDcex) fused to surface expression anchoring domain(INPNC), connected by a poly-histidine tag to be easily identified by Westernblotting. For more information, please visit our Wiki page.
Promoter and RBS: The INPNC surface expression component derives from Pseudomonas Syringae. We used parts BBa_K811005 from iGEM12_Penn. CBDcex derives from Cellulomonas fimi, and is part of an exoglucanase secreted by this organism. We used parts BBa_K1321342 from iGEM Imperial 2014 for construction.
Outer membrane porin OprF from Pseudomonas fluorescens with Plac and RBS
motB under the control of constitutive promoter J23112
This gene codes for MotB, a part of the flagellar motor. MotA and MotB constitute the stator and are required for the flow of ions and torque generation. Here we used 3 different constitutive [http://partsregistry.org/Part:BBa_J23100 Anderson promoters] from the 2006 Berkeley group to test how different levels of constitutive ''motB'' expression affect the motility of ''E. coli''.
* The part was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). * J23112 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
Outer Membrane Protein X (OmpX) with BsoBI-linker and NanoLuc
OmpX is an outer membrane protein with the C- and N-termini in the intracellulair domain. To be able to use OmpX as a scaffold, a non-natural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction with DBCO functionalized groups. The BsoBI linker is a 213 bp long flexible GGSGGS linker. Using the restriction enzyme BsoBI, the linker can become 45 bp shorter. NanoLuc is a small sized and bright luciferase. Under the addition of furimazine, the NanoLuc luciferase generates a bioluminescece emmission spectrum with a maximum around 460 nm.
The sequence is redesigned considering codon optimalization. Also restriction sites for biobricking and classical cloning are deleted. From the BsoBI linker, the coding sequecne for GGSGGS is redesigned causing that only two BsoBI restriction sites remain.
OmpX is a outer membrane protein naturally occuring in wildtype E.coli. The BsoBI linker is inspired by the article "Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Liners" by Toon H. Evers et all from 29 August 2006. NanoLuc naturally occurs in the organism Oplophorus gracilirostris and was optimalized by the company Promega.
motB under the control of constitutive promoter J23114
constitutive expression of CBDclos fused to INPNC with 6xHis tag
CBDclos fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBDclos) fused to surface expression anchoring domain(INPNC), connected by a 6xHis tag to be easily identified by Western blotting. For more information, please visit our Wiki page.
Promoter and RBS: iGEM16_Kyoto The INPNC surface expression component derives from Pseudomonas Syringae. We used parts BBa_K811005 from iGEM12_Penn. CBDclos derives from Clostridium cellulovorans, and is part of a multi-unit cellulosomes. We used parts BBa_K1321341 from iGEM Imperial 2014 for construction.
N-terminal domain of BclA protein fused to 6xHis tag
This part codes N-terminal domain of BclA protein which expressed on the cell surface connected by a 6??His tag to be easily identified by Western blotting.[1] Surface displayed passengers retain enzyme activity even when it is linked to BclA with 6?? His tag.
optimized to expression in E. coli K-12 strain
NCBI accession number CAD56880 NCBI accession number CAD56878 NCBI accession number CAD56869 NCBI accession number CAD56870
puc BA is the complete LHII transmembrane protein coding region found naturally in Rhodobacter Sphaeroides. LHII complexes form the light-harvesting antennae the funnel energy down to LHI and the reaction center. They are naturally found in the ratio of 3-6.7 LHII per LHI. Data suggests the precence of puc C,D, and E regions also contribute to the formation of a functional protein.
puc A and puc B together create the antennae complex necessary to harvest varying intensities of light. A common problem in bioreactors is the uneven distribution of light between cells nearest the light source and cells farther away from the light source. One way of combating this problem is modify the antenna size of the organism. By truncating the light-harvesting antenna, cells at the exterior of the bioreactor waste less light through Non-Photochemical Quenching, thus allowing photons to penetrate deeper and reach cells in the interior of the bioreactor. This leads to an increase in photosynthetic efficiency and productivity for the entire culture.
PCR applified from plasmid pRKCBC3 provided by Dr. Neil Hunter.
This is and engineered BioBrick derived from the BBa_K1185000 (L-form switch for ''Bacillus subtilis'')which can be used as a switch to enable the model Gram-negative bacteria ''Escherichia coli'' to switch between cell-walled rod form and wall-less L-form. The BioBrick contains homologous regions to the ftsI (homolog to pbpb in Bacillus subtilis)and murE genes to allow integration into the chromosome of E. coli via homologous recombination. Within the BioBrick there is a IPTG inducible promoter Pspac. This promoter is upstream of the murE coding sequence when integrated into the chromosome of E. coli, placing murE operon expression under the control of IPTG. The murE operon is responsible for the synthesis of enzymes involved in production of peptidoglycan synthesis. Disruption of this pathway can be utilised to down-regulate production of the cell wall. After integration of the BioBrick, the expression of murE operon is controlled by the presence, or absence, of IPTG(via the Pspacpromoter). When IPTG is present, murE operon genes are expressed and functional cell wall is produced. When IPTG is not present the cell wall is no longer produced and cells can transition to the cell wall-less L-form phenotype. The BioBrick also contains a cat gene, conferring Ampicilin resistance. This can be used to select for transformants in rod form. This BioBrick only functions in enabling switching between walled cells and wall-deficient L-forms in E. coli. We are giving opportunities for future iGEM team to work with cell wall-less E. coli. This BioBrick should work in a similar manner as the BBa_K1185000 (B. subtilis Switch BioBrick). The characterization of this BioBrick is something that future iGEM can do.
The BioBrick does not contain any illegal restriction sites. All of the sequences have been taken directly from E. coli genome so there is no need for codon optimization.
All the sequences come from the Escherichia coli K12 genomic sequence. The ftsI section in the BioBrick was taken from end of the ftsI gene 1268-1767bp (500bp). Sequence acquired through EcoCyc (EG10341). The murE section was taken from the start of the murE gene 1-500bp. Sequence from EcoCyc (EG10621). The Pspac promoter were taken from the registry designed by the 2007 Cambridge iGEM team (BBa_1746666). Ampicilin resistance marker gene was taken from the expression vector pSTAR (gb|AF047654.1|AF047654).
Added HIV protease site between OmpA and SH3 for easier release.
The HIV protease site is embedded in the protein so that a proteolytic cleavage can release the binding cassette.
OmpA is from BL21(DE3) E. Coli genomic DNA. SH3 is from human Grb2 protein. HIV protease site is from a former part.
constitutive promoter-rbs-GFP(LVA)- terminators
This part contains the coding sequence for the green fluorescent protein with a lpp signal peptide and a ompA domain