The biobricks BBa_K091146 and BBa_S03154 where used to build this biobrick
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the expresion of LasI enzyme, no terminator
YFP Producer Controlled by 3OC6HSL Receiver Device
The luxR based receiver, I13270, controls the production of the YFP. This construct has been used to characterize I13270.
position 3&5 mutated promoter lux pR-3/5 (luxR & HSL regulated)
The position 3&5 mutated promoter lux pR-3/5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-3/5 gives a lower expression of downstream genes in an autoinduction system. In another word, the lux pR-3/5 has a lower efficiency than the lux pR(BBa_R0062). Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
We have generated two point mutants by site-directed mutagenesis from the sequence of lux pR(BBa_R0062). In these mutants, the G residue of position 5 was changed to a C and the C residue of position 3 was changed to a T respectively.
Colicin-E3, its immunity protein, and colicin lysis protein is constitutively expressed strong.(under BBa_?J23101) When this device works, Colicin-E3 will be released to media, and E. coli strains which does not have immunity protein will be killed.
LuxR receiver circuit (driven by ptet) with pBad controlling the expression of aiiA. Useful for measuring the ability of aiiA (LVA+) to turn off the response of a tet driven receiver cell to exogenous HSL levels
the cheY* gene is installed in front of mRFP operon. cheY is the gene coding for cheY protein which controls flagella movement. This sequence, however, is mutated to give a mutant cheY protein which will not respond to the presence of cheZ protein. This cheY* gene is without a promoter, rbs or terminator.
the cheY* gene is without an rbs nor a terminator
3OC6HSL Sender Controlled by Lac Repressible Promoter
A lac repressible promoter driving production of an untagged LuxI enzyme which produces an AHL (3OC6HSL).
This is a two-protein generator (cI and lasR) without transcriptional terminator. Strong RBS (efficiency=0.6).
autoinducer synthetase for N-butyryl-HSL (BHL) and HHL
Debra Lin, Srini Devadas, David Gray, Ronny Krashinsky, and Chris Zheng Liu, Boopers
Autoinducer synthesis protein that produces N-butyryl-HSL which binds to RhlR, obtained from Pseudomonas aeruginosa.
Editing Check (1/28/04) (0) BB sites cleaned (1) ATG (2) TAATAA (3) Blast checks out (i.e., it's RhlI) (4) Codon changes OK (AA and tRNA usage) (5) Chassis might have conflict (w/ native autoinducer system?) (6) ssrA tag added (7) barcode added (8) Codon optimized for expression in enteric bacteria 2 silent point mutations have been included into the sequence at base 138 from A to G and at base 576 from G to C to remove internal EcoRI and PstI sites. Mutations were chosen to yield the same amino acid and codons of similar frequency in E. coli.
rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)
Lux-based Sender/Receiver Device which outputs PoPS
BioBricked version of Natural Lux system but has no terminator - allows addition of cis elements
RhlR can sense N-butyryl-HSL in the media and can activate transcription from R0071. It can be used as part of a receiver device.
Pgad - Intracellular Chloride-Sensing Cassette
Pgad is chloride-sensitive promoter which was first discovered in Lactococcus lactis1, which is a gram-positive bacterium which can live in acidic environment. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, which is constitutively expressed under the control of PgadR, is a positive regulator of Pgad coupled genes while intracellular chloride is level elevated.
Synthesized part by GenScript,all the illegal sites for RFC 10 (EcoRI, XbaI, SpeI, PstI) have been substituted by other codon producing the same amino acid in E. coli.
The gene sequence was obtained from the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG.
Hybrid promoter: HSL-LuxR activated, P22 C2 repressed
Hybrid promoter consisting of the Lux box which overlaps partly with -35 box. Binding sites for P22 C2 OR2 and OR1 are located between the -35 and -10 boxes and downstream of them respectively.
-35 box is partly composite. -10 is taken from the P22 phage PR regulatory region
Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter It produces luxI when there is enough IPTG.
The LasR binding site is from the region upstream of the LasI gene in Pseudomonas aeruginosa. The rest of the promoter is the same as BBa_K091156.
K091157 is a modified version of the pLux' promoter (BBa_K091156) containing the addition of a LasR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of AI-1 and LuxR and repressed in the presence of PAI-1 and LasR.
In order to fit the LasR binding site between the -35 and -10 regions, the last base of the Las Box was cropped and the -35 region was modified to overlap with the first two bases of the Las Box.
luxR protein generator under Plambda regulation
This device produces luxR constitutively under the strong promoter Plambda, in absence of cI repressor. Plambda can be turned off by cI repressor and luxR production cannot continue. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
HasR is the membrane-bound receptor in the Has system, a heme acquisition system originally from the Serratia marcescens. The HasR protein is involved in binding with a heme-loaded HasA hemophore to trigger downstream HasS and HasI. It has been shown that the N terminal of HasR is especially crucial for signal transduction. With the heme-loaded hasA bound to hasR, it induces a conformational change in HasR that causes the activation of HasS, an anti-sigma factor. HasS then releases HasI (sigma factor) that was initially bound to it and HasI ultimately is able to trigger expression of genes under control of pHas (part number).
The HasS gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
THIS PART DOES NOT WORK. Need to add the CI operator sites.
Complete CDS of Rat olfactory protein mRNA, clone I7.
MC31-rbs-luxI-Ter If there are microcystin, the luxI will be produced.
Pseudomonas aeruginosa rhlAB promoter
Promoter activated by RhlR in concert with C4-HSL. C4-HSL is produced by RhlI, and acts as a quorum-sensing autoinducer. This is the natural sequence taken from Pseudomonas aeruginosa. Crosstalk: this promoter is also activated at a low level by LasR with its associated HSL.
The -10 and -35 sites are labeled as identified in [Pearson97]. The part begins with the RhlR binding site, and (rather arbitrarily) ends with the first transcribed base (+1).
This biosensor is constructed to sense C-4 AHL molecules through a method different from BBa_K1157012. This time when AHL molecules are sensed, it produces Rhl receptors which initiates production of CI. Therefore, CI inhibits pCI and the expression of reporter mCherry. When compared to control group, the results will be expected to express lower fluorescence level when there is existence of AHL molecules.
The parts are built by the original biobricks provided by iGEM official, and there are no restriction enzyme sites in the inner part of the construct.
This is built by the biobricks provided by iGEM official
OMPA-golB A protein to catch gold ions. OMPA present golB outside the membrane and golB will catch gold ions.
This protein generator produces the enzyme LasI. This enzyme produces 3OC12HSL. This is the 3OC12HSL analog to part F1611.
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)
The LuxR binding site is from BBa_R0062 and the pLas' promoter is from the region upstream of the LasI gene in Pseudomonas aeruginosa.
K091146 is a modified version of the pLas' promoter (BBa_K091117) containing the addition of a luxR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of PAI-1 and LasR and repressed in the presence of AI-1 and LuxR.
The Lux box was placed adjacent to the LasR binding site with the aim of minimizing cross-talk in the presence of both PAI-1 and AI-1; since the LasR site should have a higher affinity, we hope that the binding of PAI-1 will competitively exclude the binding of AI-1.
The aptamers are from results of paper, In vitro selection of specific aptamers against microcysitin-LR. The whole riboswitches are designed.
It is a riboswitch designed by our team. We design a lot of riboswitch for microcystin, and then choose two excellent ones, MC7 and MC31, by modeling. They are riboswitches based on the stability of mRNA. The structure of them include a hammerhead ribozyme and a microcystin aptamer. Users can connect them down the promoter to use them.
We tested a lot of riboswitches by model and experiment to choose better one.
ToxR' aa's 1-210 transmembrane (inner membrane) dimerizing protein. Used in conjunction with J07007-ctx_promotor for dimerization controlled gene expression. aa's 1-210 (abridged from 1-294) removes the w.t. periplasmic region of the protein with intent to replace region with specific binding molecules.
Reporter tripped by CinR-HSL Threshold, tripped by Lux-HSL Threshold. CinR-HSL also promotes production of aiiA. This should break the thresholds and reset the system.
autoinducer synthetase for PAI from Pseudomonas aeruginosa (no LVA)
Lux pL controlled luxR with lux pR controlled GFP(lva tag).
This BioBrick system is able to notify the downstream activation of the lux R promoter of the 3OC6HSL Receiver Device.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device.
The lux p promoters originate from the quorum sensing ability of ''Vibrio fischeri''.
Promoter, Standard (luxR and HSL regulated -- lux pR)
[Note: This is the same part as R0062 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. Promoter activated by LuxR in concert with HSL 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 LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file part=BBa_R0062>Image1.gif</bb_file>" width="614" height="362"> Modified to comply with BBa_S0001: TTGACA-17N-GATACT
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr Drew Endy
The Cin R (LVA+) protein can be used to sense 3OC14HSL in the media as part of a receiver device
Autoinducer inactivation enzyme regulated by LuxR/HSL promoter
We made this construct with LuxR/HSL promoter R0062, B0034 RBS, and the C0060 aiiA gene in order to give us more control with our repressilator system. The aiiA gene will be turned on by the LuxR/HSL promoter in the presence of the LuxR activator protein complexed with the autoinducer 3-oxo-hexanoyl-HSL.
Pcons2-rbs-luxR-Ter. It achieves a constant expression of luxR, the AHL combining protein Pcons2 is BBa_J23100
MC31, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
MC31-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter It is a test of our positive feedback circuit for microcystin.
Lux Receiver (I13263 with reversed part order)
Receiver circuit similar to I13263 but with the order of the reporter and luxR generator reversed
This expression casette constitutively produces the LuxR regulatory protein that can respond to the quorum sensing molecule 3OC6. It also produces a LacI repressor with a mutation at position 12 that results in an isoleucine. The mutant repressor has been shown to have an increased affinity for its operator.
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
A part designed to emit AHL when given aTc. Uses R0040 and F1611.
Elowitz RBS. CinR CDS (no LVA tag) and terminator. Same as I0468 but no LVA tag
Prlux-rbs-rfp-rbs-luxR-Ter. It produces rfp and luxR when there is enough rfp and luxR.
Vibrio harveyi repressor protein LuxO and phosphorelay protein LuxU genes. LuxO and LuxU are regulators of luminescence in Vibrio harveyi.
LuxO and LuxU are found within the operon, therefore we have decided to keep these genes together. The natural RBS was also kept.
GenBank Accession# L26221 LuxO Protein ID# AAD12736.1 LuxU Protein ID# AAD12737.2 Jeremy A. Freeman and Bonnie L. Bassler. Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. 1999. Journal of Bacteriology. Feb 1999, p. 899-906.
V. fishceri and phage lambda (courtesy of Ron Weiss)
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 repressor, LuxR. 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.In thisversion, a binding site for cI dimer has been added: binding to this site dominantly represses transcription.
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).
unidentified/uncharacterized CinR dependent
MC7, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
MC7-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter. It is a test of our positive feedback circuit for microcystin.
single chain Antibody fragment selected to bind to fluorescein molecules. This scFv is the weakest of the three specified sequences. DNA insert in the pCT-4-4-20 (also known as pcT-302) from NheI to XhoI
Substantial Energetic Improvement with Minimal Structural Perturbation in a High Affinity Mutant Antibody
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
This gene sequence includes an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease N terminus differently instead of C terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the part of TEV protease.
CD225 (EpCAM antibody) is commonly used for the test of presence of EpCAM in the laboratories. The sequences of OmpA and TEV protease terminuses are acquired from the Registry.
GFP Producer Controlled by 3OC6HSL Receiver Device
The luxR based receiver, F2620 (formerly I13270), controls the production of GFP. The GFP protein generator is the same as that found in I7101.
GFP Producer Controlled by 3OC6HSL (without Promoter)
3OC6HSL receiver device which need a PROMOTER to control the expression of LuxR
GFP and aiiA are repressed Lux promoter GFP and aiiA induced by AHL. GFP will express if the amount of AHL exeed the capability of aiiA.
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.
Double Promoter (LuxR/HSL, positive / cI, negative)
This device senses the 3OC12HSL concentration in the media and produces output PoPS. LasR production is controlled by constitutive P(BIa). This part is the 3OC12HSL analog of F2621.
Lux pL controlled LuxR + lux pR promoter.
This part was made as a replacement for a defective version of BBa_F2621 which lacked the R0063 promoter, but was otherwise intact and functional. It shows a significantly higher response to 3OC6HSL than the incomplete BBa_F2621 (which is present in the Registry as BBa_K317037)
Was constructed by cloning BBa_R0063 in front of the incomplete BBa_F2621 (sent from the Registry in July 2011 lacking the R0063 subcomponent)
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.
Pcons2, lacI, Plac, luxI, luxR, Prlux, rfp, Ter
pGAL1 + Flag + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
This device accepts PoPS as input and produces the LuxI enzyme. This enzyme produces 3OC6HSL.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
The DNA of BBa_R0079 and BBa_F1610 biobricks where used.
This biobrick has a promoter inducible by PAI+LasR (BBa_R0079)and produces LuxI enzyme (BBa_F1610). This enzyme produces 3OC6HSL (AI).
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the lac repressible R0011.
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL 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 LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
send P-AI-1 signaling molecules out, using Mnt(or cI) protein repress the pMnt/lac( or plux/cI) hybrid promoter
Elowitz RBS. RhlR CDS (no LVA tag) and terminator. Same as I0466 but no LVA tag
Barry Canton [bcanton@mit.edu] and Anna Labno [labnoa@mit.edu]
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the tet repressible R0040. Initial characterization work has been done on this device using I13273 as a test device.
Example Data Sheets for F2620
The -35 box of R0065 has been modified to promote constitutive (LuxR+3OC6HSL-independent) gene expression. An extended -10 sequence has also been added to increase absolute transcription. The promoter still contains the operators OR1 and OR2 such that it retains its sensitivity to CI.
Plac-rbs-luxI-Ter. It produces luxI when there is enough IPTG or less lacI.
Lux pL controlled LuxR with lux pR autoinducing LuxI(lva tag)- AHL.
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. Complementary to the lux pL promoter, the lux pR promoter with a complex of luxR and 3OC6HSL will enable enlarged activation of the downstream synthetase gene, thus increase the concentration 3OC6HSL.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device. The resulting construct worked a lot better than the original F2621 - K082014 BioBrick system.
The lux p promoters and the autoinducer producing enzyme originate from the quorum sensing ability of ''Vibrio fischeri''.
PgolTS-golS-PgolB-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter It is a test of our positive feedback circuit for gold ions.
PgolTS-golS-PgolB, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
autoinducer inactivation enzyme from Bacillus; hydrolyzes acetyl homoserine lactone
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita D. Marinescu and Alexander D. Wissner-Gross
Coding region for the autoinducer inactivation enzyme A (aiiA) LVA tagged. The gene was originally isolated from Bacillus sp. 240B1 and it encodes an enzyme that catalyzes the degradation of N-acyl homoserine lactones (AHLs)--quorum sensing autoinducers.
References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 BBa_C0060 insert contains open reading frame (nucleotides 49-801) of the GeneBank sequence AF196486 followed by the LVA tag and two double stop codons inserted in the BioBrick prefix and suffix flanking regions. The original stop codon was TAG and in the present sequence it was substituted by TAATAA.
AF196486 from Bacillus sp. 240B1 putative metallohydrolase (aiiA) gene. Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000).
This device is composed of a pLuxL promoter drivinga 3OC6HSL receiver device. The output PoPS is used to produce YFP The YFP should be lightly tagged. This is the exact receiver in the MIT SBC04 synchronization device. It is identical to the previously characterized I13263 but with a tagged output device
PAI+LasR -> LasI+GFP & AI+LuxR --| LasI+GFP
The biobricks BBa_K266005 and BBa_E0840 where used to build this biobrick.
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the policistronic expresion of LasI enzyme and GFP, double terminator
autoinducer inactivation enzyme, AaiiA (+RBS)
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
sense TWO INPUTS, activation by AHL and repression by LacI.
Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a "HYBRID" of the two, we call it HYBRID PROMOTER. Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
This gene sequence includes both of EpCAM Binding Docks; BBa_K772001 and BBa_K772002. Thus, this composite part is the main component of CTC (Circulating Tumor Cell) detection system. Docks include an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C or N terminus. Hopefully, after binding to the EpCAM antigen, TEV protease domains will fix together to form the full structure in order to activate the signalling pathway. (For more information: BBa_K772003) EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequences, there are a constitutive pLac promoter and appropriate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the domain of TEV protease.
Ser H101 Ala the mid-strength of the 3 piece anti-flour escein scFv sequence set.
High Population->mRFP (pLac->LuxI; pLacIQ->luxR; pLux->mRFP;)
Quorum sensing circuit under a pLac promoter. Population turns red (mRFP) once quorum sensing kicks in.
Double terminator not used after LuxI because it is unnecessary and has the potential to make the plasmid more unstable.
Built through assembly of BBa_K084014 and BBa_J10000.
GFP and Producer Controlled by 3OC6HSL Receiver Device
The luxR based receiver, F2622 (formerly I13275 and I13280), controls the production of GFP. The GFP protein generator is the same as that found in I7101.
Lux based Sender/Receiver/Reporter system (LVA-)
Analogue of the V. Fischeri quorum-sensing system. Built using Biobrick components. Includes LuxR and LuxI under the control of their natural promoters and includes a YFP output device on the same transcript as LuxI (Same as I13264 but LuxI has no LVA tag)
Rbs-luxI(scarless) It is an improvement of luxI(BBa_C0061) by adding a rbs.
It is an improvement of luxI(BBa_C0061) by adding a rbs.
single chain Antibody fragment selected to bind to fluorescein molecules. This scFv is the strongest of the three specified sequences.
CinR, CinL and glucose controlled promotor
This promotor is controlled by cinr, cinl and extrcellular glucose concentration. High extracellular glucose concentration will inhibit this promotor through the action of phosphorylated OmpR. Phosphorylated OmpR will bind the OmpR binding site of the promotor and block the movement of RNA polymerase. In contrast, the combined action of CinR and CinL will turn on the promotor. But only when the extracellular glucose level is low so OmpR dephosphorylate and dissociate from its binding site will this promotor become active. In essence, this promotor function as an AND gate, it will turn on if and only if the extracellular glucose level is low and there are some functional CinR and CinL protein inside the cell.
The key to the function of the promotor is the inhibitory ability of OmpR binding site. To ensure maximum affinity with phosphorylated OmpR, we use all three OmpR binding site.s.
The rpfG gene from Xanthomonas campestris
This is the coding sequence for the RpfG(regulation of pathogenicity factor G) protein. It is the regulator in the two-component signal transduction system of Xanthomonas campestris pv. campestris that is activated by the sensor kinase RpfC(Bba_K1315002) in the presence of DSF(diffusible signal factor).
The sequence had 1 EcoRI site that was 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.
LasR and 3OC12HSL regulated promoter with CI Operator 1 (binding site for lambda cI repressor)
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
Produces LasR protein which can sense AI-1 in the media and activate transcription from R0078, the right hand Lux promoter
a fusion protein consisting of E.coli endogenous SOS system repressor LexA and fungus N.crass photosensor protein VVD with LexA carrying mutation at position 40-42 and VVD carrying mutation N56K, C71V and I135M. This part serves as an ultra sensitive photoreceptor(which can sense light as weak as moonlight) and will induce a light-dependent repression of genes with their promoter containing mutated 408 form of SOS box.
LexA carrying mutation at position 40-42 and VVD carrying mutation N56K, C71V and I135M enable the sensor recognize 408 form of SOS box and with enhanced performance.
Barnase and GFP is co-expressed by luxR/AHL complex.
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the LuxR controllable R0063.
Colicin-E3, its immunity protein, and colisin lysis protein is constitutively expressed.(under BBa_J23110) When this device works, Colicin-E3 is released to media, and kill E. coli strains which does not have immunity protein.
BioBricked version of the natural Lux system with order reversed
Places both LuxI and LuxR under the control of their natural promoters but reverses the order relative to I13211
A slightly different sequence than the original; the coverage has been shifted. It should function the same way the original was intended, left repression by hsl-bound LuxR and right activation by hsl-bound LuxR.
The biobricks BBa_K266006 and BBa_K266000 where used to build this biobrick
Complex Quorum sensing circuit that receives the signal of PAI+LasR and AI+LuxR to control the production of LuxI and LasI enzymes.
This part involves the -10 binding site, the -35 binding site, and the twenty nucleotides between that constitute the lux box. With this part, LuxR functions as a acyl-homoserine lactone-dependent repressor. LuxR resonds to the HSL produced by LuxI, N-(3-oxohexanoyl)-HSL. The Lux box is positioned such that it partially overlaps the consensus -35 and -10 hexamers of an RNA polymerase binding site.
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
lasI: autoinducer synthetase for PAI from Pseudomonas aeruginosa. Strong RBS (efficiency=0.6).
this part comes from LuxR promoter and cI lambda promoter.
This hybrid promoter is designed to respond to activation by LuxR when it is bound to AHL and to repression by the phage lambda cI repressor. The repression needs to win over activation.
This part is based on the idea of replacing the region of the Lux promoter downstream of the -35 with the OR2 cI half-operator, added LuxR-10 region. Based on what we have found up to now, promoters which are constructed just by inserting OR1/2 downstream of Lux are all very leaky.
Generates lasI in response to aTc input.
This AHL receiver contains mutant LuxR. it can express GFP 0.1x AHL conc. compared wild type.
Site direct mutagenesis -> Ligation with Biobrick
Colicin-E3 cleave 16SrRNA at a specific site. This activity is inhibited by immunity protein for Colicin-E3(BBa_). When lysis protein for Colicin-E3(BBa_) is co-expressed, Colicin-E3 is emited to environment and kill other strain. Colicin is a type of bacteriocin produced by Escherichia coli.
PoPS->PoPS amplifier (CinR-based, HSL driven)
CinR protein generator upstream of pCin. Useful device for cell-cell signaling. Expected to require HSL for maximum PoPS out.
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
This part is a combination of the two parts K091155 and K091144.
These two parts contain parts that are in the registry and DNA built from oligos.
luxR protein generator under Plambda regulation (TERM-)
luxR protein generator without transcriptional terminator. This generator is under Plambda repressible promoter. Strong RBS (efficiency=0.6).
Plux-rbs-CI-terminator-plambda P(O-R12)-rbs-cheZ
This is a part in which the expression of cheZ is repressed by increasing AHL concentration in the environment in the presence of luxR protein. In other circumstances the cheZ gene is constitutively expressed. The cheZ gene lacks a terminator.
OmpR binding site. When position behind a promotor, it will turns that promotor into a OmpR controlled promotor. OmpR will function as an inhibitor of that promotor.
This part was derived from BBa_R0082 by PCR.
Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter. It produces rfp, luxI and luxR when there is enough luxR and AHL.
LuxR and aiiA express constantly. AiiA inactivate AHL as signaling molecule. If AHL concentration exceed the capacity of aiiA, GFP expresses.
This part is the one of the responding systems of the receiver device of our Photocommunication system
We used ncbi when we couldn't find sequence
EPIC Luciferase: BBa_K325100, Cambridge 2010 pOmpC: BBa_R0082
An enhanced lacI protein generator, inhibits Plac perfectly
This is an enhanced lacI generator. Induced point mutations shifted the structure so it can stop transmitting more perfectly.
Several point mutations were made while this works the best.
A graduate essay published in Zhejiang University, China. Sequence is open to the public.
The chimeric GPCR consists of the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the c. elegans OR odr-10. It can be functionally expressed in S. cerevisiae, activating the yeast endogenous MAPK pathway upon binding to the ligand diacetyl.
position 5 mutated promoter lux pR-5 (luxR & HSL regulated)
The position 5 mutated promoter lux pR-5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-5 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-5 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
By PCR method the G residue of position 5 was changed to a C based on the sequence of the promoter lux pR(BBa_R0062).
P2 promoter in agr operon from S. aureus
The section of the sequence constituting the P2 promoter is subject to rather more debate than the sequences of the coding regions. [http://jb.asm.org/cgi/content/full/186/22/7549 Koenig, Robbin L., Ray, Jessica L., Maleki, Soheila J., Smeltzer, Mark S., Hurlburt, Barry K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region J. Bacteriol. 2004 186: 7549-7555] shows the areas of the sequence to which phosphorylated AgrA binds while L Rao, R K Karls, and M J Betley: In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. Bacteriol. 1995 May; 177(10): 2609???2614. shows the transcription start sites; we took the overall extent of the promoter region from that labelled in Morfeldt, E., K. Tegmark, and S. Arvidson. 1996. Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. 21:1227???1237. (not available online).
The agr P2 operon is an autocatalytic sensory transduction system in Staphylococcus aureus. P2 promoter regulates the synthesis of AgrB (a transmembrane protein) and AgrD (precursor of AIP autoinducing peptide). AIP binds to AgrC, a membrane binding receptor and it phosphorylates AgrA. The phosphorylated AgrA increases the expression level from P2 promoter.
Codon usage has been optimised for E. coli (which also seems to give good results for B. subtilis
A part designed to emit AHL when given aTc. Uses R0040 and F1610.
lasR activator from P. aeruginosa PAO1(no LVA)
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.
Promoter (luxR & HSL regulated -- lux pL)
The lux cassette of V. fischeri contains a left and a right promoter. The left promoter gives weak constitutive expression of downstream genes.This expression is down-regulated by the action of the Lux repressor, LuxR. 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 from Pr, repressing transcription from Pl
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below.Includes most of Lux reulatory region, including the LuxR binding site which activates the right promoter. A putative LuxR autorepression binding site is also identified adjacent to the -10 site of the right promoter. This 2nd site has 55% identity with the first site. Putative inverted repeats (of size 18-27 bp) also exist between these two sites (not marked above), which may represent binding sites for other regulatory proteins. <img src="<bb_file>Image01.gif</bb_file>" width="614" height="362"> Unspecified.
LasR/LasI Inducible & RHLR/RHLI repressible Promoter
Rust L. et. al., "Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Journal of Bacteriology, 1996.
This part is similar to BBa_R0079 but has an added binding site for RHLR/RHLI in between the -35 and -10 region. Thus this promoter is induced by LasR/LasI but repressed by RHLR/RHLI binding.
The natural spacer between the -35 and -10 is 14bp and the natural O1 site for RHLR/RHLI is 20bp long. There was not much over lap of the -35 or the -10 with this sequence, so it was nessicary to chop off some of the binding site sequence in order to maintain the correct spacing and preserve the -35 and -10 sequence. The binding site sequence is missing a tcc on is 5' end and a t on its 3' end.
this hybrid promoter is designed to be consecutively off.It combines with plasR(R0079) and lambda cI OR1(see C0051).Adding P-AI-1 sould activate the promoter, while repressed by lambda cI.
"CI binding of the OR region. It binds OR1 with highest affinity, OR2 with next highest, and OR3 with lowest"(http://www.nonanone.com/qw/files/Biology%204450/test2_lecture8,9.txt)
Prlux-rbs-rfp-rbs-luxI-Ter. It produces rfp and luxI when there is enough luxR an AHL.
Receiver circuit. LuxR production is controlled by R0011. Output device is untagged YFP. Because R0011 is stronger than R0040, the switch point of this receiver should be lower than I13273. Built from the following parts - R0011.A340620.E0430
The pLux' promoter is a modified version of BBa_R0062 containing the -35 region TTTAAT in place of TTTACG. Transcriptional activation is expected to occur in the presence of LuxR and AI-1, just as with R0062. The modified -35 region was designed to serve as a control for the evaluation of BBa_K091156, the pLux+ Las- promoter.
This promoter is identical to pLux+ Las- with the exception of the Las box's absence
Composite part based on the A340620 PoPS amplifier. pLuxL promoter driving luxR-based HSL receiver. pLux-R is driving a YFP output device.
position 3 mutated promoter lux pR-3 (luxR & HSL regulated)
The position 3 mutated promoter lux pR-3 is based on promoter lux pR (BBa_R0062).
The position 3 mutated promoter lux pR-3 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 3 mutated promoter lux pR-3 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-3 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
In the promoter lux pR's sequence, the C residue of position 3 was changed to a T using PCR method.
autoinducer inactivation enzyme aiiA (no LVA)
This device receives AI-1, produces LasR, and reports its production with GFP
comes from existing parts in the Registry (S03966 + S03956)
Anti-fluorescein scFv (4M5.3, 15 point mutations) with transcriptional terminator.
cinI codes for an autoinducer synthetase which utilizes SAM to make O3-C14:1-HSL. In complex with O3-C14:1-HSL, CinR (BBa_C0077) 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: tga -> tAaTAA
rhlR repressor/activator from P. aeruginosa PA3477 (+LVA)
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
Prlux-rbs-rfp-Ter. It produces rfp when there is enough luxR and AHL.
aiiA (LVA-) protein generator driven by ptet
Produces aiiA protein from the tet promoter. The aiiA is untagged and should be capable of degrading any intracellular AHL.
aiiA (LVA-) protein generator driven by plac
Produces aiiA protein from the lac promoter. The aiiA is untagged and should be capable of degrading any intracellular AHL.
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.
Anti-fluorescein scFv (4-4-20, no mutations) with transcriptional terminator.
Outputs varying levels of aiiA as part of HSL/aiiA measurements. aiiA is untagged. Similar to I13204
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.
LuxR receiver circuit with an inducible promoter controlling aiiA production downstream. Useful to measure the ability of aiiA (LVA+) to turn off a pLuxL driven receiver circuit in the presence of exogenous HSL
luxI protein synthesizes 3OC6HSL, which binds to LuxR. Strong RBS (efficiency=0.6).
The HasR gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
This part was obtained by taking the reverse complement of R0062, ordering appropriate oligos, then annealing and extending them.
This promoter is the right promoter in the lux cassette of Vibrio Fischeri. There is weak constitutive expression of downstream genes. Expression of downstream genes can be up-regulated by the action of the LuxR activator protein in complex with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein can form a complex with two molecules of the AHL. This causes a conformational change in the protein, allowing it to bind to a site on the lux promoter and induce transcription.
Genomic sequence of Pseudomonas aeruginosa
Promoter driving the rhlI gene in Pseudomonas aeruginosa. Activated by the RhlR+C4HSL. Also likely activated by LasR+3OC12HSL
lsrR coding gene with ribosome binding site B0034.
Promoter driving the lasI gene in Pseudomonas aeruginosa. Activated by LasR+3OC12HSL and not RhlR+C4HSL (Seed et al., 1995) ~4x stronger than the lasB promoter (BBa_R0079; Seed et al., 1995). Switch point ~0.1nM 3OC12HSL (Seed et al., 1995)
Promoter sequence cropped to 4nt upstream of Las box according to example of Whiteley and Greenberg, J. Bac, 2001
Lux based Sender/Receiver/Reporter system
Analogue of the V. Fischeri quorum-sensing system. Built using Biobrick components. Includes LuxR and LuxI under the control of their natural promoters and includes a YFP output device on the same transcript as LuxI
LuxS is a synthase for the AI-2 quorum sensing system. This protein is involved in the production of small molecules that are readily absorbed by multiple species of bacteria. LuxS can be used as part of a positive feedback loop in cell to cell signaling.
Nucleotide position 54 changed from A to T in order to destroy a PstI site; this is a sense mutation Also changed stop codon from TAG to TAA (recommended in Registry Help)
The LuxS sequence was pulled from E. coli MG1655 by predesigned primers.
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
Binding region for LasR protein (positive regulation)
This gene sequence includes an EpCAM (Epithelial p? Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease N terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease C terminus differently instead of N terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
This device senses the C4HSL concentration in the media and produces output PoPS. RhlR production is controlled by constitutive P(BIa). This part is the C4HSL analog of F2621.
Luminesensor repressible LacIM generator
LacI mutant driven by luminesensor repressible SulA promoter in 408 form.
B0034, CDS of LacI mutant and B0015 were connected directly to SulA promoter in 408 form.
Sequene of the LacI mutant is from Partregistry.
Synthesizes 3OC6HSL, which binds to LuxR. 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 repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
An LVA tail (sequence: AANDENYALVA) was added to increase protein degradation. .
autoinducer synthetase for PAI from Pseudomonas aeruginosa
coding region for lasI protein, which produces the chemical signal AI-1
The BCAM0228 Response Regulator from Burkholderia
This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderia spp.
The source of this part is Burkholderia cenocepacia J2315 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxI-Ter It is a positive feedback plasmid using luxI as key to feedback. Introducing IPTG will produce rfp and luxI, and luxI will work back to enhance production of rfp.
Luminesensor repressible RecA408 promoter+CheZ
CheZ coding sequence ligated to the Luminesensor repressible recA408 promoter
CheZ is got from E.Coli strain MG1655 by colony PCR
accept AHL signal to produce RoPS and could also produce mRFP
could be easily connect to a protein coding region with a terminator
autoinducer synthetase for N-butyryl-HSL (BHL) and HHL (no LVA)
MC7-rbs-luxI-Ter If there are microcystin, the luxI will be produced.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 V01146 .
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
aiiA Protein Generator (LVA+) Controlled by a tet Repressible Promoter
Outputs varying levels of aiiA as part of HSL/aiiA measurements. Subpart of I13205 Formerly known as I13204
PgolTS-golS-PgolB-rbs-luxI-Ter If there are gold ions, the luxI will be produced.
This is the coding sequence for AgrD, the gene encoding the peptide precursor to Autoinducing Peptide, AIP. The AgrD protein product is co-translationally exported, cleaved, and circularized via the AgrB membrane exporter. It then binds to the extracellular domain of AgrC initiating a signalling cascade within the cell.
We wanted to use the coding sequence without the E. coli RBS present in BBa_I746001 and so PCRed this part out. It can be used with any RBS, including gram-positive compatible ones such as BBa_K090505.
Odr-10(diacetyl)GPCR with optimized codon
Junqi kuang, Xihao liao and Jianhui gong
Odr-10(diacetyl)GPCR encodes a diacetyl-sensing G-protein coupled receptor, originating from the C. elegans. If BBa_K1072010 was transformed into the yeast, the pheromone receptors will be actived and coupled to a heterotrimeric G protein, Odr-10. pathway is composedof Gpa1p (Gα), Ste4p (Gβ) and Ste18p (Gγ) subunits.The Gβγ particle transmits the signal to a MAP kinase module and finally follow a inducible promotor FUS1, which will link a reproter, such as GFP, His3 or LacZ.
As discription of most paper, RNA of Odr-10 be used as plate and transform into receptor cells to express member protein successfully.
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.
Biobricked version of the natural Lux quorum sensing system
This device includes a positive feedback loop where LuxI production produces 3OC6HSL that binds to LuxR and activates transcription from R0062 producing more LuxI. There is no output from this device, it must be combined with another device, controlled by R0062 to produce output.
LasR and 3OC12HSL regulated promoter with a single Tet Operator (binding site for TetR repressor)
LasR coding region and a double terminator
The biobricks BBa_C0079 and BBa_B0015 where used for this biobrick.
Anti-fluorescein scFv (one mutation, S101A) with transcriptional terminator.
Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
Transcriptional terminator consisting of a 64 bp stem-loop.
Tlanslational unit of Colicin Lysis Protein (for colicin-E3)
Colicin lysis protein allows colicins to be released. The mechanism how the colicin lysis protein allows colicin release has not been fully elucidated, but it is sure that this protein raise membrane permiability and cause quasilysis.[1] Colicins are a cytotoxins which are released to environment and kill other related strains.
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
pGAL1 + Flag + Brho + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
PoPS in -> luxI out. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
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
3OC6HSL Receiver Device with Inducible Control of LuxR and a YFP Output device
3OC6HSL receiver with a YFP output device. Same as I13034, but with a TetR regulator under the control of Plac (R0010)
37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
Lux pL controlled luxR with lux pR controlled AHL degrading enzyme AiiA(lva tag).
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. If 3OC6HSL is present in the cell, the autoinducer inactivation enzyme A (''aiiA''), with an LVA Tag, will inactivate the 3OC6HSL autoinducer molecules.
PoPS->PoPS Amplifier Controlled by 3OC6HSL
LuxR protein generator upstream of pLuxR. Useful device for cell-cell signaling. Expected to require HSL for maximum PoPS out. Putting this device under the control of a promoter produces a receiver device
LuxR Protein Generator Controlled by the Left Lux Promoter
This is the BioBrick version of the natural LuxR expression device.
The sequence of the LasI upstream regulatory region is available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=176640
This pLas' promoter contains the regulatory region upstream of the LasI gene in Pseudomonas aeruginosa. K091117 includes a LasR binding site as well as a -10 region modified from CATAAA to TATAAA to reflect our pLasXOR promoter design. Activation is expected to occur in the presence of the autoinducer PAI-1 and the LasR protein. The pLas' promoter serves as a control for the evaluation of the pLasXOR promoter, part BBa_K091118.
To minimize leaky transcription, a secondary transcriptional start site was excluded from the design.
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.
This is a cheZ gene that can be induced by luxR protein under high AHL concentration. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement.
composited from E. Coli cheZ gene and lux promoter
LuxPQ from Vibrio harveyi LuxP is a ABC-type sugar transport system periplasmic protein (Protein ID = YP_001447483.1). LuxQ is a histidine kinase (Protein ID = YP_001447484.1).
LuxP and LuxQ are found in the operon, thus we decided to keep them together. We kept natural RBS in front of LuxP, as well as the intrinsic terminator. Silent mutations were performed on a EcoRI site located within LuxP, EcorI site within LuxQ, and XbaI site within LuxQ.
GenBank Accession: NC_009784 Vibrio harveyi ATCC BAA-1116 chromosome II, REGION: 579654..580751. Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM. Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell. 2006 Sep 22;126(6):1095-108. Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM.
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
this part could accept AHLs and also could produce mRFP easily to detect
ligate the mRFP producer and AHL acceptor together
Conversion of the iGEM parts registry to SBOL2.1