PkdpF[-15, T>C] - GFP generator
This part consists codon optimised ChnR (BBa_K1946000) from acinetobacter sp. operated by Tet operator and promoter region of ChnB (BBa_K1946001) followed by sfGFP (BBa_I746916). It is shown that ChnR activates ChnB protein in the presence of cyclohexanone. This sensor generates sfGFP in the presence of cyclohexanone.
ChnR gene synthesised with respect to Uniprot amino acid sequence. Codon optimised for E. Coli k12
ChnR protein and pChnB promoter from acinetobacter sp.
Coding Sequence of IscA2 pigeon homologue (codon-optimized for humans)
The gene Iron-Sulfur cluster assembly 2 (IscA2) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to add the start codon since this second isoform did not have methionine built in.
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005502393.1), and codon-optimized for humans with IDT's tools.
fdeR (Naringenin binding protein) and fde operon regulatory domain
Sascha Hein, Niklas Hummel, Sebastian Barthel, Thomas Dohmen
fdeR - Naringenin binding protein We modified the naringenin biosensor from Siedler et al. by elimination of one EcoRI restriction site and by changing the native ribosomal binding site into the strong RBS Bba_B0034. Based on this reporter protein and 3 different fluorescence proteins, we designed 3 new biosensors for in vivo detection and determination of naringenin. The natural source of FdeR is Herbaspirillum seropedicae. You can use our reporter for measuring naringenin concentrations in your samples. Depending on which fluorophor you want to detect, you can use one of three biosensors: GFP K1497020 mKate (DsRed variant) K1497021 CFP K1497022 You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use FdeR Biobrick K1497019 and clone your parts of interest (without RBS!) behind the device.
AlkR is a transcriptional regulator belonging to AraC/Xyls family, which could detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. It is said that AlkR is the only bioreporter that is able to detect alkane with carbon chain length greater than C18.
AlkR with strong promoter and terminator.
PkdpF[-15, T>A] - GFP generator
Charlotte Bunne, Mariam Harmouche, Stefan Holderbach, Anna Huhn and Jakob Kreft (in alphabetical order)
This is a part for the precise quantification of UV-radiation or radioactive radiation in E. coli (recA+) strains, i.e. BL21(DE3). It consits of a recB Promoter (part BBa_K862003) fused to a LacZ reporter cloned in front of a double terminator (part BBa_K173004).
The part was first constructed in plasmid backbone pSB1AK3 and then subcloned into pSB1C3 for submission to the registry.
The part was constructed by standard cloning from parts BBa_K862003 (precB) and BBa_K173004 (LacZ).
The universal stress protein A is a response of Escherichia coli cells to growth arrest, and its lacking generates cells with defective growth.This genetic element is dependent to sigma-70 factor. Its regulation is done through the concentration of a specific stationary phase allormone, guanosine-5'-diphosphate-3'-diphosphate (ppGp), as described elsewhere. The ppGp activate the transcription of downstream elements through a positive regulation of the β-subunit of RNA polymerase. In this sense, the PuspA element is considered a stationary phase promoter. However, its behavior is like a constitutive transcription promotion, becoming higher in stress situations. Diverse conditions make the Escherichia coli cell enter to stress state, like heat shocks, starvation, osmotic stress, ultraviolet light and other conditions. In these situations, the RNA polimerase sigma factors (ropS) trigger the expression of chaperones and other stress protector molecules, in order to help the cell survive. In our project we have used this promoter to trigger the expression of limonene synthase after an osmotic shock. Other uses of this part includes the environmental stress detectors making using a reporter system coupled to this promoter.
The original promoter site was shortened and the last three nucleotides were changed to restriction site adjustment.
We have drawn the sequence for an improved PuspA promoter from an analysis of 400bp upstream the Universal Protein A (UspA) gene in E. coli K12 MG16555 genome. After design, the DNA template of part was synthesized by IDT and was amplified from a G-block and assembled using 3A assembly method.
Strong reporter device optimized for Pichia pastoris, activated by methanol
This device is composed by three parts. The first part is a modified promoter pAOX, it was found in a library where it showed more than 60% stronger activation when compared with the wild-type, as reported by Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A: Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 2008, 36:e76. The second part is the kozak sequence for Pichia pastoris, and the third is a Red fluorescent protein condon optimized for expression in Pichia pastoris. This device was created to express a reporter gene in the presence of ethanol, and is inactivated by ethanol, when combined with the modified Mxr1 (BBa_K1078000), it is not inactivated by ethanol. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normal content of 10 to 60% ethanol). This will allow government to make high-throughput screening of ethanol drinks tainted will methanol. The modified Mxr1 is able to activate the pAOX promoter, in ethanol, glycerol and methanol. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normal content of 10 to 60% ethanol). This will allow government to make high-throughput screening of ethanol drinks tainted will methanol.
After synthesis, in order to build our sensor the part was introduced in pPIC9K plasmid to allow its genomic recombination into the Pichia pastoris genome, substituting the original Mxr1 found in the yeast. This part does not have a single biobrick version with the Mx1p because both must be inserted in different genome locations at the Pichia pastoris
he part was obtained by synthesis, the modified Mxr1p sequence was found in Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A: Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 2008, 36:e76
FRET based ATP biosensor for expression in Aspergillus niger
Consists of cfp-yfp fret pair that responds to changes in ATP concentration
It has illegal restriction sites for the standard 10 assembly. The cds of the part is flanked by NdeI and NotI restriction sites.
NsrR - NO(x) sensitive pYear, pNirk repressor protein
This part contains the NsrR (NO(x) sensitive pYear, pNirk repressor protein) encoding region. Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. PyeaR is regulated by the NsrR repressor protein. Nitric oxide binds to the NsrR protein and relieves the repression on PyeaR. As a result, any genes that are downstream of PyeaR are expressed. This part is also related with BBa_K2056002, the promoter region of the NirK protein, because NsrR activates the transcription of NirK under high nitrite conditions. Due to these properties, we can use this part as a sensor for oxides of nitrogen.
Because the part Bba_K1682011 was not available from the registry, we had to get it synthesized. We had to codon optimized this part to conform to assembly standards and the requirements for synthesis.
This part has been designed after the part Bba_K1682011 in the Registry of Standard Biological Parts.
phoApis a phosphate-responsive promoter. In high concentration of phosphate, it will be repressed while in low phosphate concentration, it will be activated.
phoAp is native in E. coli
This part responds to the presence of toluene, m-, p- or o-xylenes and produces a green fluorescent signal. The transcriptional regulatory protein XylR recognizes these organic molecules and activates pu promoter which in turn will lead to the synthesis of sfGFP.
XylR (BBa_K1946005) in this composite part was codon optimized by us for E.coli K12.
The source of XylR (BBa_K1946005) can be found at GenBank: AAB59162.1 . pu promoter (BBa_K1946006) was synthetically synthesized from GeneWiz based on Pseudomonas putida TOL plasmid. sfGFP sequence can be accessed in the registry at BBa_I746916.
AlkR is a transcriptional regulator belonging to AraC/Xyls family, which could detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. It is said that AlkR is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. The mechanism is that alkanes are presumably recognized by AlkR regulatory protein and their interaction triggered a conformation change of AlkR dimers which isomerizes the promoter-RNAP complex and lead to the activation of alkM gene. AlkR regulatory protein includes C-terminal DNA-binding domain(CTD) for promoter binding and N-terminal domain(NTD) for inducer recognition. Here we use a constitutive promoter for AlkR gene and in case of interference, we choose a long and strong terminator.
We use a constitutive promoter for AlkR gene and we choose a long and strong terminator to prevent interference.
This is a part for the precise quantification of UV-radiation or radioactive radiation in E. coli (recA+) strains, i.e. BL21(DE3). It consits of a SulA Promoter (part BBa_K518010) fused to a LacZ reporter cloned in front of a double terminator (part BBa_K173004).
The part was constructed by standard cloning from parts BBa_K518010 (pSulA) and part BBa_K173004 (LacZ reporter).
Blue-Yellow fluorescent protein toggle switch using pLac and OmpC promoter.
Ryan Kawalerski, Sarah Heacox, Jonathan Krog
Alkane Selector for intracellular alkane production
Naringenin sensor (FdeR) with CFP as reporter
We modified the naringenin biosensor from Siedler et al. by elimination of one EcoRI restriction site and by changing the native ribosomal binding site into the strong RBS Bba_B0034. Based on this reporter protein and 3 different fluorescence proteins, we designed 3 new biosensors for in vivo detection and determination of naringenin. The natural source of FdeR is Herbaspirillum seropedicae. You can use our reporter for measuring naringenin concentrations in your samples. Depending on which fluorophor you want to detect, you can use one of three biosensors: GFP fluorescence K1497020 mKate (DsRed variant) fluorescence K1497021 CFP fluorescence K1497022
This is a part for the precise quantification of UV-radiation or radioactive radiation in E. coli (rec+) strains, i.e. BL21(DE3). It consits of a recA Promoter (part BBa_J22106) fused to a LacZ reporter cloned in front of a double terminator (part BBa_K173004).
The part was constructed by standard cloning from parts BBa_J22106 (pRecA) and part BBa_K173004 (LacZ reporter).
FRET based ATP biosensor cds, codon optimized for Aspergillus niger
Shreyans Chordia (shreyanschordia90@gmail.com)
A FRET based biosensor that will measure the ratio of ATP and ADP.
The rpfC gene from Xanthomonas campestris
This is the coding sequence for the RpfC(regulation of pathogenicity factor C) protein. It is the sensor in the two-component signal transduction of Xcc that activates the regulator RpfG(Bba_K1315003) in the presence of DSF.
The sequence had 1 EcoRI site and 3 PstI sites that were all mutated out by site-directed mutagenesis.
The source of this part is ''Xanthomonas campestris pv. campestris''(Xcc) genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
AlkR is a transcriptional regulator belonging to AraC/Xyls family, which could detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. It is said that AlkR is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. The mechanism is that Alkane were presumably recognized by AlkR regulatory protein and their interaction triggered a conformation change of AlkR dimers which isomerizes the promoter-RNAP complex and lead to the activation of alkM gene. AlkR regulatory protein includes C-terminal DNA-binding domain(CTD) for promoter binding and N-terminal domain(NTD) for inducer recognition.
We choise a constitutive promoter for AlkR gene and in case of interference, we use a long and strong terminator.
PkdpF[-15, T>G] - GFP generator
The recB promoter sequence was taken for the E. coli MG1655 genome sequence (http://ecoliwiki.net/colipedia/index.php/recB:Gene). We assumed the main promoter region to be located from -70 to -1 bp upstream the recB start codon. Therefore this sequence was synthesized and cloned on an oligo basis. We will not submit the physical DNA of this part, but we provide the oligo-sequences you can use for synthesizing and cloning this part in front of any EcoRI/XbaI precut reporter part. RecB_fw: aattcgcggccgcttctagagCCTGAAGGCTGGAAAGTGTGGGAGAACGTCAGCGCGTTGCAGCAAACAATGCCCCTGATGAGTGAAAAGAc RecB_rev: ctaggTCTTTTCACTCATCAGGGGCATTGTTTGCTGCAACGCGCTGACGTTCTCCCACACTTTCCAGCCTTCAGGctctagaagcggccgcg
the sequence was ordered on a synthetic oligo basis and cloned in front of EcoRI/XbaI reporter parts.
E. coli MG1655 genome: http://ecoliwiki.net/colipedia/index.php/recB:Gene bp -70 to -1 before recB coding sequence.
PphoA promoter is a phosphate-responsive promoter. In high concentration of phosphate, it will be repressed while in low phosphate concentration, it will be activated.
An acid inducible promoter of the acid shock response (asr) gene, extracted from E. coli K12. This part can be used to generate pH based response in any biological system.
The sequence provided in literature indicated a consensus sequence for the Pho Box just before the transcription start site
This part was extracted from the genome of E. coli K12
Coding Sequence of IscA1 pigeon homologue (codon-optimized for humans)
Michael Becich, Thomas Lau, Sreyas Misra
The gene Iron-Sulfur cluster assembly 1 (IscA1) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to remove the start codon in order to co-express a fluorescent protein, and modify two BbsII restriction enzyme sites by altering the third base pair of codons (wobble effect) at bp locations 75 (C->G) and 309 (A->G).
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005508102.1), and codon-optimized for humans with IDT's tools.
NsrR protein is a nitrate sensitive transcriptional repressor. When there is nitrate, it will be oxidized to nitric oxide by nitrate oxidase, which in turns relieve the repression of NsrR on PyeaR promoter.
Naringenin sensor (FdeR) with mKate as reporter