AgrA protein coding sequence. AgrA is the main component of S.aureus agr quorum sensing system.
Staphylococcus aureus AgrA, the transcriptional component of a quorum sensing system and global regulator of virulence that up-regulates secreted virulence factors and down-regulates cell wall-associated proteins, can bind in both the P2 and P3 promoter regions of the agr locus. 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.
As a AIP sensor system, agrA should work with agrC.
lasR activator from P. aeruginosa PAO1(+LVA)
coding region for lasR protein, which accepts chemical signal AI-1 (made by lasI protein)
Mutated 480 from g to a to remove PstI site
Genbank: NC_002516 (www.ncbi.nlm.nih.gov)
This is a MoClo converted version of BBa_C0079
This is a MoClo converted version of BBa_C0079, which is the coding region for the lasR protein, which accepts chemical signal AI-1 (made by lasI protein). It has two MoClo fusion sites flanking it.
We had to design the proper MoClo fusion sites to flank this part.
In this portion of the design, we aim to achieve population control by combining a cell-signaling system (luxR/luxI) and suicidal gene for controlling the number of E.coli. The population controlling function in the engineered E.coli can warn us of high-density E. coli congregations by emitting fluorescent proteins. Green fluorescent light means the Cry circuit is functional and the population density is under control, while red fluorescent gene signals E.coli overpopulation.
(I) Strand C (BBa_ K332021) 1. Strand C is synthesized from three fragments ??? (1) Ptet (2) RBS+LuxR (3) RBS+LuxI+double ter sites. 2. All of them are in the form of plasmids, so we digest them by appropriate restriction enzymes. 3. We first combine the last two fragments by ligation. The procedure is as follows: (1)Add the respective digestion products, vector plasmid, buffer, ligase to ddwater (2)React at 16??C for 30 minutes (3)Inactivate the ligase by incubation it at 80??C for 20 minutes (4)Store the products at 4??C 4. The products of ligation are then transformed into competent cells. The procedure is as follows: (1)2??l plasmids(ligation products) are added to 33??l competent cells (2)Shake gently for one second, and cool it on ice (3)Incubate it in 42??C water bath for 45 seconds (4)Spread the liquid to plate which corresponding antibiotics have been added to (5)37??C overnight 5. The next day we do a colony PCR for the transformed cells and have the PCR products electrophoresis to make sure that the two fragments have been combined correctly. 6. Now we separate the plasmids(RBS+LuxR+RBS+LuxI+double ter sites) from E. Coli, and digest it with restriction enzyme. 7. The first fragment (Ptet) is then combined with the above product by ligation, and transformed into competent cells by the same we have done early. 8. Because the length of Ptet to the remain segment is relatively too short, we cannot confirm whether they have combined correctly by just do an electrophoresis of the colony PCR products. If the electrophoresis result seems like that they have combined, we then send the product to sequencing.
1. LuxI protein (BBa_C0061) is the synthase that converts S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When the concentration of AHL reaches a threshold level, it binds to the N-terminal domain of a LuxR protein. Then the LuxR/AHL complex binds the Lux box within PtetR promoter (BBa_C0040), which then activates suicidal gene expression. 2. LuxR (BBa_C0062) is transcribed continuously by the constitutive promoter (BBa_J23106), and later works in conjunction with AHL. This design can control the concentration of LuxR protein in a steady phase, and the concentration of AHL/LuxR complex is only affected by the concentration of LuxI protein (BBa_C0061).
Sequence for sigma 32 transcription factor
This part is a correction to [http://parts.igem.org/Part:BBa_M36888 BBa_M36888] which has an illegal PstI site which makes it incompatible with BioBricks / RFC10. iGEM Teams can use this alternative sequence which contains none of the BioBrick restriction enzyme sites. Amino acid No. 202, Glysine has been mutated from CAG to CAA to remove the PstI site. The remainder of the sequence remains unchanged.
This part was checked for BioBrick restriction enzyme sites which would otherwise make it unsuitable for use by iGEM teams.
This part is a correction to [http://parts.igem.org/Part:BBa_M36888 BBa_M36888].
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - IFP[1]-CpxR + IFP[2]-CpxR (BBa_K1286009) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
All orientations were made, so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used for each of the fused proteins, and an RBS was put before each one of them.
The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a VP64 fused to its C terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription.
CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
This is the ''virG'' response regulator from the virA/G receptor system. The virA/G receptor system is used by ''Agrobacterium tumefaciens'' to detect phenolic substances secreted by plants. In presence of these substances, ''virG'' is activated by the ''virA'' receptor and induces the transcription of genes under the control of a ''vir'' promotor. Normally, ''virG'' needs the ''rpoA'' RNA polymerase subunit from ''A. tumefaciens'' to work in ''Escherichia coli'' but this ''virG'' BioBrick is mutated, so works with the ''rpoA'' subunit from ''E. coli''.
synthesized by Mr. Gene, so optimated codon usage, removal of illegal restriction sites
vir promoter + sensitivity tuner + luciferase
This part combines a mutated ''virG'' response regulator from the VirA/G receptor system under the control of a constitutive promoter with a sensitivity tuner before a luciferase gene under the control of a ''vir'' promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
sensitivity tuner to enhance the luciferase signal
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''vir'' promoter from ''Agrobacterium tumefaciens'' C58, TI-plasmid
The Pseydomonas quinolone signal (PQS) as well as its precursor 2-heptyl-4-quinolone (HHQ)are very important in mechanisms controlling virulence in Pseudomonas aeruginosa. P. aeruginosa has a key role in Cystic Fibrosis (CF). PQS can initiate signalling for various processes like virulence, motility, and biofilm formation. A good number of these processes are facilitated by PqsR, which is a transcriptional factor. An activated PqsR will go to the nucleus of the cells and activate the respective genes. http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector.
The source of this part is the genome of Pseudomonas aeruginosa PAO1 provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[2]-CpxR. Biobrick BBa_K1486014 has IFP[1]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486013 is CpxR-IFP[2] (with an arabinose promoter).
Both orientations were made, as well as for IFP[1] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
This part promotes the constitutive expression of the curli cluster (csgD), proteins involved in cell-cell adhesion.
This part regulates curli expression within the cell via transcriptional activation. The construct can be repressed in the presence of the TetR repressor.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[2]. Biobrick BBa_K1486012 has CpxR-IFP[1] with an arabinose promoter. This construct was made in both orientations: BBa_K1486015 is IFP[2]-CpxR (with an arabinose promoter).
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" ! colspan="2" style="background:#FFBF00;"|VP64 |- |'''Function''' |transcription activator |- |'''RFC standard''' |[http://parts.igem.org/Help:Assembly_standard_10 RFC 10] |- |'''Backbone''' |pSB1C3 |- |'''Organism''' |''synthetic construct'' |- |'''Source''' |synthetic construct |- |'''Submitted by''' |[http://2016.igem.org/Team:NEU-China NEU-China 2016] |} Tetrameric VP16 transcription activator domain
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
In complex with HSL, LuxR binds to the Lux promoter, activating transcription from Pr <bb_part>BBa_R0062</bb_part>, and repressing transcription from Pl <bb_part>BBa_R0063</bb_part>. The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux activator, LuxR complexed to HSL. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
2 silent point mutants were introduced in the coding sequence to remove internal XbaI and PstI sites. Mutation sites were chosen to replace codons commonly used in E. coli with codons used at a similar frequency.
V. fischeri <genbank>AF170104</genbank>
Celia Chenebault, Camille Soucies, Benjamin Piot
The Pr promoter is a constitutive promoter driving the transcription of the XylR gene, coding for the XylR protein. The XylR protein, a transcriptional regulatory protein for the Pu promoter, is activated by aromatic hydrocarbons that carry a methyl group (like toluene and xylene). In our biosensor the Pu promoter allows the transcription of the bioluminescent reporter gene GLuc, coding for the Gaussia luciferase. When this enzyme reacts with its coelenterazine substrate it emits luminescence.
Site directed mutagenesis to overcome the mutation in the start codon of XylR
IDT synthesis in 3 parts that were then assembled
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[1]. Biobrick BBa_K1486013 has CpxR-IFP[2] with an arabinose promoter. This construct was made in both orientations: BBa_K1486014 is IFP[1]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
This part contains: - a mutated ''virG'' response regulator from the virA/G receptor system from ''Agrobacterium tumefaciens'' which works in ''Escherichia coli'' without an ''rpoA'' gene from ''A. tumefaciens'' - a kanamycin resistance under the control of a ''vir'' promotor This device is used to screen ''virA'' receptor mutants. The ''virA'' receptor is mutated in the BBa_K389010 BioBrick. The better the receptor detects a substance, the more ''virG'' is phosphorylated and the stronger the kanamycin resistance from this BioBrick is expressed. The BioBricks BBa_K389010 and BBa_K389011 have to be used together in one cell.
medium strong constitutive promotor, terminator to keep basal expression of kanamycin resistance low
synthetic ''virG'' gene, ''vir'' promotor from ''A. tumefaciens'' C58, partsregistry
appY coding sequence encoding a DNA-binding transcriptional activator
This is the coding sequence of appY from Escherichia coli JM109. It encodes a transcriptional regulator related to anaerobic energy metabolism. Overexpression of the gene has been reported to result in enhanced lycopene production by E. coli (Kang et al., 2005).
Encodes a transcription activator for the right handed Lux promotor. Two molecules of acyl-homoserine lactones (AHL) form a complex with two molecules of the LuxR protein. This complex then binds to a palyndromic site on the lux promoter, increasing its transcription rate. LuxI, LuxR and the right lux promoter are all derived from V. fischeri.
Biobrick BBa_C0061 still contained a Barcode sequence which was deleted. For our own use, 9 bp were exchanged to create unique restriction sites and to optimize DNA folding.
Sequence is originating from V. fischeri. Here, the sequence from BBa_I0462 was used and optimized.
Plasmid containing LuxO D47A (pJAF544) was obtained from Bassler lab (Freeman and Bassler, 1999).
Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
This part combines a mutated virG response regulator from the VirA/G receptor system under the control of a constitutive promoter with a sensitivity tuner before a luciferase gene under the control of a vir promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
global DNA-binding transcriptional dual regulator
PCR using primers with biobrick prefix and surfix, and then cloned into pSB1AC3.
XylS senses benzoate and its substitutes, it is the transcriptional activation factor that activates the expression of meta-operon for benzoate degradation on TOL plasmid pWW0 in Pseudomonas putida. This part contains XylS coding sequence with terminator. To complete the sensor, please add promoter before XylS coding sequence.
Molecular cloning to add terminator to XylS coding sequence.
XylS comes from Pseudomonas putida, sequence found through Genbank: http://www.ncbi.nlm.nih.gov/nuccore/M10143.1
lasR activator from P. aeruginosa PAO1(no LVA)
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
This is a coding sequence of heat shock protein from E.coli
this is a coding sequence of heat shock protein from E.coli. It regulate heat shock proteins .....
HSP15 derived from genome E.coli K12 http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=G7743-MONOMER
toxicity-gene activator from Vibrio cholerae
shortened BBa_J07008. Typical ToxR-periplasm-region subsitutions are done @ aa 211. explanation of ToxR domains @ http://model.mit.edu/igem/images/d/d7/Kolmar95.pdf ELIMINATE XbaI SITE PLZZZZZ
T7 amber expression system in Bacillus subtilis.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxR-Ter. It is a positive feedback plasmid using luxR as key to feedback. Introducing IPTG will produce rfp and luxR, and luxR will work back to enhance production of rfp.
Pcons2, lacI, Plac, luxI, luxR, Prlux, rfp, Ter
Autoinducer synthetase for acyl-homoserine lactones (AHL): LuxI synthesizes AHL (3OC6HSL). Two molecules of AHL form a complex with two molecules of the LuxR protein. This complex then binds to a palyndromic site on the lux promoter, increasing its transcription rate. LuxI, LuxR and the right lux promoter are all derived from V. fischeri. In absence of AHL, the right Lux promotor only gives weak constitutive expression of downstream genes. Any gene you would like to be controlled by AHL and thus cell density can be cloned after this complex.
For the ease of cloning, the sequence was devided in multiple gBlocks. Therefor, there is a mistake in the composite part sequence since the scar is only partly present.
The Lux operon originates from V. Fischeri.
rcsA is a gene responsible for regulating capsule synthesis. It has also been shown to induce lambda lysogens into the lytic cycle.
Periplasm transport visualization device
VirA/G signaling system without read-out
This BioBrick is a virA/G signaling system without read-out. It contains an unmutated ''virA'' gene and a ''virG'' gene that works without ''rpoA'' from ''A. tumefaciens'' and a ''vir'' promoter.
This BioBrick can be used to control any gene with a ''vir'' promoter
''A. tumefaciens'' C58 TI-plasmid, partsregistry, Mr. Gene
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[1]-CpxR. Biobrick BBa_K1486015 has IFP[2]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486012 is CpxR-IFP[1] (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put between the promoter and the cds.
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
This part is activator of proteins responsible for acid resistance in E.coli. GadE is transcription factor of Gad operon and Gad operon express Glutamate Dependent Acid Resistance genes gadA, gadB, gadC.
CpxR is a transciption factor that dimerizes and becomes active when phosphorylated by the transmembrane histidine kinase CpxA. CpxA in turn becomes active when the bacterial cell is submitted to different kinds of shock (pH, temperature, pressure, protein misfolding in the inter-membrane space...).
When making composite parts with CpxR that are supposed to dimerize, we had to take care about the positioning of the other elements regarding the parallel dimerization of CpxR.
The part has been extracted from the E coli genome.
Prlux-rbs-rfp-rbs-luxR-Ter. It produces rfp and luxR when there is enough rfp and luxR.
Coding sequence for PAX3, a regulatory transcription factor in H. sapiens
Brett Fuller, Austin Jones, Jeremy Gerbig, Sindhu Adhikari, Coby Turner
Contains coding sequence for PAX3, a transcription factor which binds to the Mitf promoter. When expressed in a system with the Mitf promoter present, will induce production of target gene.
Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter. It produces rfp, luxI and luxR when there is enough luxR and AHL.
araC arabinose operon regulatory protein (repressor/activator) from E. coli (+LVA)
coding region for the araC gene, used to make araC protein for use in positive or negative regulation of TIPs output (see also R0080, R0081)
GenBank: NC_002655 (www.ncbi.nlm.nih.gov)
The clp gene from Xanthomonas campestris
c-di-GMP Repressed DNA Binding Protein (Clp) Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris
The sequence had 1 PstI site that was mutated out by site-directed mutagenesis.
The source of this part is Xanthomonas campestris pv. campestris 8004 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
LasR coding region and a double terminator
The biobricks BBa_C0079 and BBa_B0015 where used for this biobrick.
XylR coding device-GFP coding device with Pu
XylR coding device (BBa_K2023002) and GFP coding device with Pu (BBa_K2023016)
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter It is a positive feedback plasmid using luxI and luxR as key to feedback. Introducing IPTG will produce rfp, luxI and luxR, and luxI together with luxR will work back to enhance production of rfp.
Originally VP16 is a protein from the herpes simplex virus. It activates the transcription of the late genes of this virus by the RNA-polymerase II of the eucaryotic host. In artificial systems VP16 is used in fusion with a DNA-binding domain. This DNA-binding domain is specific for a bait sequence. So the selective expression of genes with a promoter containing this bait sequence can be achieved.
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick. <br\n> For more information about this issus, see Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." DSpace http://hdl.handle.net/1721.1/32535 (2006).
We have constructed our BioBrick from the plasmid xxx, which was available in our hosting lab.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-Ter. It is a control plasmid for positive feedback circuit. It will produce rfp when there is enough IPTG.
Pcons2, lacI, Plac, luxI, Pcons2, luxR, Prlux, rfp, Ter
TALE that induces the BS3 promoter. The part is compatible with the GoldenGate MoClo Assembly Standard (free from BsaI and BpiI recognition sequences) Codon-optimised for expression in dicotyledenous plants
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants
CII bacteriophage lambda trascriptional activator
CII is an important part of bacteriaphage lambda operon.
CII is an transcriptional activator,it can
Tetrameric VP16 transc. activator domain
rhlR repressor/activator from P. aeruginosa PA3477 (+LVA)
Debra Lin, Srini Devadas, David Gray, Ronny Krashinsky, and Chris Zheng Liu, Boopers
Transcriptional regulator, in complex with N-butyryl-HSL, RhlR binds to the Rhl promoter
(1/28/04 Edit Jamboree) (0) No BB sites (1) ATG (2) TAATAA (3) Blast checks out (4) No codon changes (5) Maybe chassis compatability (check by experiment) (6) Codon usage not optimal (but looks OK) (7) ssrA tag added
This part codes for the transcriptional regulatory protein XylR from Pseudomonas putida. It activates the TOL plasmid xyl operons in the presence of toluene, m-, p- and o-xylene.
The sequence was codon optimized for E.coli K12.
The source translation sequence can be accessed at GenBank: AAB59162.1
Shamik Mascharak, Justo Caballero and Sibel Sayiner
The rpoH gene codes for sigma 32 (alternatively sigma H, RpoH) transcription factor, a component of RNA polymerase sigma 32 holoenzyme. This 32-kD stress factor belongs to the sigma-70 factor family and is involved in intracellular temperature regulation, initiating bacterial transcription of various chaperone proteins and proteases in response to sudden increases in ambient temperature. The gene is currently classified as essential, due to the fact that rpoH deletion mutants fail to grow at standard incubation temperatures. This part may be employed in heat shock-to-PoPs sensors by coupling expression of the rpoH gene to action of sigma 32-inducible promoters such as the htpG heat-shock promoter (part#: BBa_J45405). Transcriptional activation by rpoH transcripts is possible both within and without the sensor genetic circuit, as sigma factors can diffuse around the nucleus.
Designs incorporating this part into a feedback amplification loop must beware of likely rpoH-deletion mutants. This is due to overproduction of sigma 32 under leaky promoters such as htpG (BBa_J45405). Less deletion mutants in E. coli are noted when plasmids containing rpoH are cloned at higher temperature. Consider two facts before using this part. First, rpoH transcript activity is reduced under excess heat shock protein production. As such, feedback loops using rpoH should employ weaker bicistronic ribosome binding sites so as to optimize heat-shock response. Second, sigma 32 is turned over rapidly, with a half-life under 1 minute.
The particular part described here is specific to the genome of W3110 strain E. coli.
CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. The protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. The protein to be a dimer in the absence of CO. The product, CooA, is 28% identical (51% similar) to CRP(cAMP receptor protein) and 18% identical (45% similar) to FNR(fumavate nitrate reduction) from Escherichia coli. Inactive Fe(II) CooA structure adapted from that of the strain with PDB identification no. 1FT9. The protein consists of two monomers, shaded differently, which dimerize along the central C-helices of adjacent effector-binding domains. The solved structure is asymmetric, in which one monomer contains fused C- and D-helices. Nonetheless, both F-helices that interact with DNA in a sequence-specific manner are buried from the surface in the structure. The 4/5 loop is noted and so are the Pro2 and His77 heme Fe(II) ligands.
The sequence information was acquired from NCBI and physical DNA was synthesized from GENEART. Classical cloning strategies(restriction digestion) were used to produce biobricks.
Hwan Youn, Robert L. Kerby, Mary Conrad, et al. 2004. Functionally Critical Elements of CooA-Related CO Sensors. J. Bacteriol. 186(5):1320-1329. doi:10.1128/JB.186.5.1320-1329.2004.
The part contains CpxR with an upstream arabinose-inducible promoter.
The arabinose had to be upstream of the CpxR. Also, the sequence had to be tested because of a mutation, and it works perfectly.
The CpxR comes from the genome of E coli, and the arabinose promoter comes from a biobrick.
LuxO D47E, Vibrio harveyi is a constitutively active mutant of LuxO
The plasmid containing LuxOD47E (pJAF822) was obtained from Bassler lab (Freeman and Bassler, 1999). Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
>gi|156972381:c2983472-2982069 Vibrio harveyi ATCC BAA-1116 chromosome I, complete sequence Nucleotide number 141 has been mutated from T to G, thus bases 139-141 have been changed from GAT (Aspartic acid, D) to GAG (Glutamine, E). Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
To edit. Pense ?? rajouter les linkers, je ne sais pas s'ils sont de igem ou s'il faut ajouter la s??quence ?? la registry. Check pour tous tes biobricks et dis-moi, please.
Pcons2-rbs-luxR-Ter. It achieves a constant expression of luxR, the AHL combining protein Pcons2 is BBa_J23100
This part is the twin of the non-compatible biobrick BBa_K1486008 rendered compatible by mutating the two illegal restriction sites. However, all experiments were run on the aforementioned biobrick.
Site-directed mutagenesis was used to remove the restriction sites in the sequence.
http://www.addgene.org/browse/article/8177/ which was then mutated by us.
Yichong wang, Ruichen, Xihao liao and Junjie zhang
BBa_K1072002 is the generator fo NADH dehydrogenase which is an enzyme following chemical reaction: NADH + H+ acceptor \rightleftharpoons NAD+ + reduced acceptor
cinR activator from Rhizobium leguminosarum (+LVA)
In complex with O3-C14:1-HSL (made by BBa_C0076), CinR binds to the Cin promoter (BBa_R0077) and activates transcription.
The regulatory locus cinRI in Rhizobium leguminosarum conrols a network of quorum-sensing loci Lithgow, JK; Wilkinson, A; Hardman, A; Rodelas, B; Wisniewski-Dye, F; Williams, P; Downie, AJ MOL. MICROBIOLOGY 37(1): 81-97, 2000 Change log: original STOP: tag -> taATAA
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''vir'' promoter from Agrobacterium tumefaciens C58, TI-plasmid
Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter It produces luxI when there is enough IPTG.
This part codes for ChnR protein which is a Xyls/AraC-type transcriptional activator from Acinetobacter sp. It responds to the presence of cyclohexanone by inducing expression of ChnB protein. Iwaki H, Hasegawa Y, Teraoka M, Tokuyama T, Bergeron H, Lau PC. Identification of a transcriptional activator (ChnR) and a 6-oxohexanoate dehydrogenase (ChnE) in the cyclohexanol catabolic pathway in Acinetobacter sp. Strain NCIMB 9871 and localization of the genes that encode them. Appl Environ Microbiol. 1999;65(11):5158-62. Steigedal, Magnus and Svein Valla. "The Acinetobacter Sp. Chnb Promoter Together With Its Cognate Positive Regulator Chnr Is An Attractive New Candidate For Metabolic Engineering Applications In Bacteria". Metabolic Engineering 10.2 (2008): 121-129. Web.
The source of this part is amino acid sequence from GenBank: BAA86295.1