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
PgolTS-golS-PgolB-tetR-Ter If there exist gold ions, tetR will be produced.
Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter. It produces lacI when there is enough AHL.
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
mig1 is a repressor targeting the promotor of suc2.
Sequence and information is based on the Saccharomyces Genome Database, see http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=mig1
Fusion protein of SpyCatcher and RPD3 in an expression cassette
Maltose binding protein fused to Zif268-GCN4.
PgolTS-golS-PgolB-tetR-Ter-PtetO-rfp-Ter-Plac-tetR-Ter It is part of our test of suicide circuit by gold ions.
PgolTS-golS-PgolB, tetR, PtetO, rfp, Plac, Ter
TUP1 - repressor domain for transcription factors in yeast
This BioBrick is the N-terminal repression domain of the yeast general repressor TUP1.<br\n> In artificial systems TUP1 is used in fusion with a DNA-binding domain. This DNA-binding domain is specific for a bait sequence. So the selective repressionn 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).
Prlux-rbs-rfp-rbs-luxR-Ter. It produces rfp and luxR when there is enough rfp and luxR.
rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)
Amplified from MG1655 (accession NC_000913) chromosome using primers listed below (annealing sequences are underlined) lexA_BB_F TTCGAATTCGCGGCCGCTTCTAGATGAAAGCGTTAACGGCCAG lexA_BB_R CGGCCGCTACTAGTATTATTACAGCCAGTCGCCGTT
This part contains a LacI repressor and a terminator (BBa_C0012 + BBa_B0014). The LacI repressor repressed pLac promoter from the E.coli lactose operon. So LacI repressor is really reliable to use when we want to control a gene expression by using pLac promoter. In fact when LacI is not expressed, pLac is activated, and when there is an expression of LacI, pLac is inhibited. We can easily design a system to induce the transcription of LacI and creates a failed feedback mechanism for pLac inhibition. History: In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside).
When we design the bio-brick it was impossible to obtain a functional bio-brick by using the plasmid SB1AK3. But it's worked with a pSB1A2.
Part:BBa_C0012, Designed by Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton Group: Registry Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16).
mnt repressor (strong) from Salmonella phage P22 (+LVA)
wild type dimeric repressor from enterobacteriophage p22 with dissociation constant of 2.5*10^-12. sequence was translated with the E.Coli K12 codon usage table, and nucleotides 95-97 was changed from AAT to AAC to avoid the cut site GAATTC (94-99). Identification of Functionally Important Residues in the DNA Binding Region of the MNT Repressor Kendall, KL; Sauer, RT The Journal of Biological Chemistry, 264(23):13706-13710, 1989
the Gifsy-1 phage lysogenic repressor gogR
GogR expression is controlled by the pRM promoter and for repression gogR can bind to three different operator sites OR1, OR2, OR3 on either prophage, which also overlap with the pR promoter, so pRM and pR share certain operator sites.
There were problems expressing gogR with a string RBS on a high copy number plasmid.
Gifsy-1 is a lambdoid prophage present in Salmonella enterica
the Gifsy-2 phage lysogenic repressor gtgR
GtgR is homologous to GogR and its expression is controlled by the pR promoter and for repression gtgR can bind to three different operator sites OR1, OR2, OR3 on either prophage, which also overlap with the pR promoter, so pRM and pR share certain operator sites.
No problems expressing gtgR with a strong RBS from a high copy number plasmid.
Gifsy-1 is a lambdoid prophage present in Salmonella enterica. Gene sequence obtained from Sebastien Lemire, DTU.
lacI IS mutant (IPTG unresponsive) T276F
Short description: The lacI IS mutant is almost identical to the lacI transcriptional regulator except for the difference that it is not able to bind IPTG or allolactose due to a mutation; it therefore can not be activated by induction with these substances. Since it recognizes the same motif in the lac promotor region, it strongly represses transcription of all genes regulated by promotors with lacI binding site even if IPTG or allolactose are present. It can be used to terminate the expression of proteins under lac control if IPTG can not be removed from the cell rapidly. Detailed description: Expression of the lac operon in E. coli is tightly controlled by lacI, a protein, which binds to a repressor binding site within the promotor and disables transcription by obscuring the promotor region. When bound to DNA, lacI is in the tetrameric form, which consists of two dimers interacting at the end distal from the DNA binding site. Upon binding of allolactose or IPTG, the tetramer breaks down into two dimers and the affinity for the repressor binding site is greatly reduced; the lacI IPTG complex will diffuse away from the repressor binding site, leaving the promotor accessible. As a result of decades of genetic and structural studies, the function of lacI is now understood on the molecular level (1, 2). Mutational experiments have identified residues, which abolish IPTG response upon mutation (3). Furthermore, the x-ray crystal structure of lacI with bound IPTG has allowed the identification of residues that interact with IPTG and which are promising targets for mutagenesis (1). We decided to mutate residues R197 and T276, which are located in the IPTG binding groove, contact IPTG and have been shown to produce the lacI IS mutation in previous genetic experiments. Since a quantitative study of the strength of inhibition by different lacI IS mutants has to our knowledge not been published so far, we decided to generate a set of eight mutated lacIs, in which we replaced either R197 with alanine or phenylalanine or T276 with alanine or phenylalanine or both in all possible combinations. lacIIS-1: R197A lacIIS-2: R197F lacIIS-3: T276A lacIIS-4: T276F lacIIS-5: R197A T276A lacIIS-6: R197A T276F lacIIS-7: R197F T276A lacIIS-8: R197F T276F References: (1) Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247-54. (2) Friedman, A. M., Fischmann, T. O., and Steitz, T. A. (1995) Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science 268, 1721-7. (3) Suckow, J., Markiewicz, P., Kleina, L. G., Miller, J., Kisters-Woike, B., and Muller-Hill, B. (1996) Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J Mol Biol 261, 509-23.
During site-directed mutagenesis, the codons to be mutated were replaced with the most highly utilized codons in E. coli to prevent complications from the use of rare codons. The lacI IS sequences were analyzed for BioBrick restriction sites within the coding sequence to ensure their compatibility.
The lacI IS mutants presented derive from the BioBrick holding lacI by itself (C0012). Site-directed mutagenesis was performed by PCR, subsequent DpnI digest and transformation. The following primers were used: R197F forward CGGCGCGTCTGTTTCTGGCTGGCTG R197A forward CGGCGCGTCTGGCGCTGGCTGGCTG R197F reverse CAGCCAGCCAGAAACAGACGCGCCG R197A reverse CAGCCAGCCAGCGCCAGACGCGCCG T276F forward GGATACGACGATTTTGAAGACAGCTC T276A forward GGATACGACGATGCGGAAGACAGCTC T276F reverse GAGCTGTCTTCAAAATCGTCGTATCC T276A reverse GAGCTGTCTTCCGCATCGTCGTATCC All lacI IS holding BioBricks were verified by sequencing.
(correct) temperature sensitive promoter
marR(G95S) carries a G>A mutation at position 284 which gives a amino acid change G95S. Has been mutated from BBa_K864200.
Coding region for the HK022 bacteriophage cI protein. cI binds to the HK022 pR regulator (BBa_R0050). It represses transcription of the protein encoded by the sequence 3' to the pR region. This coding sequence does not contain a RBS.
References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. Derived from <genbank>STHK022N</genbank>. Response from John Little (Arizona) regarding the start of HK022 cI From: <jlittle@email.arizona.edu> Date: Tue Jan 21, 2003 4:39:21 PM US/Eastern To: "Drew Endy" <endy@MIT.EDU> Subject: RE: hk022 cI start (naive question)? Hello Drew and Michael, I seriously doubt that the extra 27 aa are part of CI. It doesn't make sense in terms of the biology, for sure. In any case, the protein we characterized starts where Carlson and Little stated; as I recall, oR3 partially overlaps the start of cI. I have a vague memory of some possibly interesting biology, having to do with multicopy plasmids. I don't recall the findings themselves. Anyway, one possible explanation was that this extra N-terminal addition was made (e.g. from the pRE promoter, or from a message that arose from transcription around the entire plasmid), making a protein with altered functions. We never followed it up. Good luck John -- Original Message -- Date: Sun, 19 Jan 2003 15:26:26 -0500 Subject: hk022 cI start (naive question)? Cc: elowitm@rockefeller.edu To: jlittle@u.arizona.edu From: Drew Endy <endy@MIT.EDU> Hi John, I'm sitting here with Michael Elowitz and we're working through the sequence for HK022 cI. We noticed that the annotation from NC_002166 includes an "extra" 81 base pairs upstream of what we thought of as the actual start. It looks like this extra DNA extends into the PR regulatory region. We're wondering what's going on here? I'm guessing this is well documented somewhere. Thanks for any pointers/info! Drew It is unknown whether there is cross-talk between this repressor and Lambda cI regulatory region, 434 cI regulatory region and P22 regulatory region.
Human phosphodiesterase 5A gene silencing device NO.1
This device is uesd for silencing the human phosphodiesterase 5A (PDE5A) gene.A U6 promoter driving a designed, synthetic shRNA-like miRNA followed by the terminator. PDE5A is a cGMP-binding, cGMP-specific phosphodiesterase, a member of the cyclic nucleotide phosphodiesterase family. This phosphodiesterase specifically hydrolyzes cGMP to 5'-GMP. It is involved in the regulation of intracellular concentrations of cyclic nucleotides and is important for smooth muscle relaxation in the cardiovascular system. We designed 3 silencing device and test their function at the same time.Here is the another two device :BBa_K1720004,BBaK172005 This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa to detect cGMP level. The results are as follow:
1. Kukreja RC, Salloum FN, Das A: Cyclic Guanosine Monophosphate Signaling and Phosphodiesterase-5 Inhibitors in Cardioprotection. Journal Of the American College Of Cardiology 2012, 59(22):1921-1927. 2. Li L, Haider HK, Wang L, Lu G, Ashraf M: Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction. American Journal Of Physiology-Heart And Circulatory Physiology 2012, 302(10):H2112-H2121. 3. Unwalla HJ, Li HT, Bahner I, Li MJ, Kohn D, Rossi JJ: Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein. Journal of virology 2006, 80(4):1863-1873.
We designed the shRNA according to the homology segment sequence of PDE5A from NCBI
TP901 cI repressor from phage TP901-1 (+LVA)
Dimeric repressor from bacteriophage TP901-1 with dissociation constant of 2*10^-9. Sequence was translated with the E.Coli K12 codon usage table, and nucleotides 84-86 was changed from TTC to TTT to avoid the cut site GAATTC (81-86). Bronsted, L., Hammer, K., Johansen, A.H., 2003. Identification of operator sites of the CI repressor of the phage TP901-1: evolutionary link to the other phages. Virology 311, 144-156. cI sequence slightly different from part C0051. NCBI locus: NC_001416
LacI repressor (temperature-sensitive mut 241) (+LVA)
Mutated from BBa_C0012 Repressor, LacI (LVA+) to be temperature sensitive. Mutation from Yabuta, 1995: Ala241->Thr (G(730)CG->ACG)
lacI IS mutant (IPTG unresponsive) R197A T276F
lacI IS mutant (IPTG unresponsive) Short description: The lacI IS mutant is almost identical to the lacI transcriptional regulator except for the difference that it is not able to bind IPTG or allolactose due to a mutation; it therefore can not be activated by induction with these substances. Since it recognizes the same motif in the lac promotor region, it strongly represses transcription of all genes regulated by promotors with lacI binding site even if IPTG or allolactose are present. It can be used to terminate the expression of proteins under lac control if IPTG can not be removed from the cell rapidly. Detailed description: Expression of the lac operon in E. coli is tightly controlled by lacI, a protein, which binds to a repressor binding site within the promotor and disables transcription by obscuring the promotor region. When bound to DNA, lacI is in the tetrameric form, which consists of two dimers interacting at the end distal from the DNA binding site. Upon binding of allolactose or IPTG, the tetramer breaks down into two dimers and the affinity for the repressor binding site is greatly reduced; the lacI IPTG complex will diffuse away from the repressor binding site, leaving the promotor accessible. As a result of decades of genetic and structural studies, the function of lacI is now understood on the molecular level (1, 2). Mutational experiments have identified residues, which abolish IPTG response upon mutation (3). Furthermore, the x-ray crystal structure of lacI with bound IPTG has allowed the identification of residues that interact with IPTG and which are promising targets for mutagenesis (1). We decided to mutate residues R197 and T276, which are located in the IPTG binding groove, contact IPTG and have been shown to produce the lacI IS mutation in previous genetic experiments. Since a quantitative study of the strength of inhibition by different lacI IS mutants has to our knowledge not been published so far, we decided to generate a set of eight mutated lacIs, in which we replaced either R197 with alanine or phenylalanine or T276 with alanine or phenylalanine or both in all possible combinations. lacIIS-1: R197A lacIIS-2: R197F lacIIS-3: T276A lacIIS-4: T276F lacIIS-5: R197A T276A lacIIS-6: R197A T276F lacIIS-7: R197F T276A lacIIS-8: R197F T276F References: (1) Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247-54. (2) Friedman, A. M., Fischmann, T. O., and Steitz, T. A. (1995) Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science 268, 1721-7. (3) Suckow, J., Markiewicz, P., Kleina, L. G., Miller, J., Kisters-Woike, B., and Muller-Hill, B. (1996) Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J Mol Biol 261, 509-23.
Rbs-cI(scarless) It is an improvement of cI(BBa_C0051) by adding a rbs.
It is an improvement of cI(BBa_C0051) by adding a rbs.
Pseudomonas putida, phiR73 phage, E.coli
Plasmid2 in detector: Prlux-rbs-cI-Ter-PcI-rbs-lacI-Ter It is a low level detection plasmid. It produces rfp when AHL is enough.
Rbs-lacI(scarless) It is an improvement of lacI by adding a rbs.
It is an improvement of lacI by adding a rbs.
This is a reverse version of BBa_C0012, the lacIq repressor cds.
Standard biobrick prefix and suffix were used. Since PCR was used, DNA sequence verification was needed - no mutations were found.
PCR was used to amplify C0012 using primers that switched the biobrick prefix and suffix.
This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. This part also produces LacI protein as an oscillator which results that as LacI protein is synthesized, the promoter wil be inhibited until the presence of IPTG. The inhibition will decrease the production of LacI which leads to the synthesis of this inhibitor. >In the absence of LacI protein and CAP protein, this part promotes transcription. >In the presence of LacI protein and CAP protein, this part inhibits transcription. >LacI can be inhibited by IPTG. >LacI is coded by BBa_C0010
Note that this part is not working unless IPTG is present in the media.
Prlux-rbs-cI-Ter. It produces cI when there is enough luxR and AHL.
Tetracycline Repressor with Consitutive Promoter BBa_J23106
A transcriptional regulator TetR (BBa_C0040) is driven by constitutive promoter family members (BBa-J23103, BBa-J23109, BBa-J23116, and BBa-J23106), which can be used to tune the expression level of constitutively expressed TetR.
The SsrA site at the end of BBa_C0040 is not present in our design.
The source of this part is based on the parts in the registry distribution
c2 repressor from Salmonella phage P22 (+LVA)
The P22 c2 repressor protein coding sequence is a 720 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the P22 c2 regulatory sequence, BBa_R0053. The sequence contains a LVA tag for faster degredation.
References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000) References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000)
Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter. It produces rfp, luxI and luxR when there is enough luxR and AHL.
Human phosphodiesterase 5A gene silencing device NO.3
A DNA cassette that undergo homologous recombination into E.coli genome that contains homologous regions at each end, Kanamicin resistance gene (KanR) and death operon containing Lambda holin and lysozime genes and controlled by a Lambda cI regulated promoter
part is synthetic. holin and lysozime originates from lambda bacteriophage. the homology regions were taken from E.coli BL21 genome
Zif23-GCN4 engineered repressor (+LVA, C2000 codon-optimized for E.coli)
Variant of Zif23-GCN4 (see BBa_C2000). CDS has been optimized (via VNTI) for expression in enteric bacteria. ssrA tag (LVA) added.
This protein should form a dimer and bind a specific operator site, thus repressing transcription.
Protein Engineering Gods from the Pabo Lab
penI repressor from Bacillus licheniformis (+LVA)
Since penI is being ported from Bacillus Licheniformis, the sequence was changed within the coding region to reflect codon usage in E.Coli K12. Codons were mapped to the codon of the same usage rank in E.Coli.
Rbs-tetR(scarless) It is an improvement of tetR(BBa_C0040) by adding a rbs.
It is an improvement of tetR(BBa_C0040) by adding a 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
Plac-rbs-tetR-Ter. It produces tetR when there is enough IPTG or less lacI.
Prlux-rbs-lacI-Ter. It worked out an expression of lacI in the presence of luxR and AHL.
LexA - a repressor for LexA-regulated SOS promoters -
Escherichia coli and other organisms have a gene cluster activated in case of emergency, which is called "SOS genes". It is well-documented that in E. coli, most of those SOS genes are repressed by LexA protein. When the cell is exposed to excess stress, RecA is up-produced, which promotes the self-degradation of LexA. As a result, many LexA-regulated genes become expressed. Combined with RecA () and SOS promoters (for instance, ), a stress-responsive gene-expression circuit was assembled. See ().
Cloned de novo from Escherichia coli JM109 strain.
Strong RBS + LacI repressor + double terminator
This part contains a strong ribosome binding site, the LacI repressor and a double terminator (BBa_B0030 + BBa_C0012 + BBa_B0014). This brick comes from the BBa_K292005. The LacI repressor represses pLac promoter from the E.coli lactose operon. So LacI repressor and this bio-brick are really reliable to use when we want to control a gene expression by using pLac promoter. In fact when LacI is not expressed, pLac is activated, and when there is an expression of LacI, pLac is inhibited. We can easily design a system to induce the transcription of LacI by using another operon as the tetracycline operon and creates a failed feedback mechanism for pLac inhibition and so, for gene expression inhibition. The LacI (BBa_K292006) already contains a strong RBS and a double terminator, so this brick is ready to use! History: In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside).
Part:BBa_B0030, Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab Part:BBa_C0012, Designed by Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton Group: Registry Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16)
LacI (Lva-, N-terminal deletion) regulatory protein
LacI is a regulatory protein responsible for the repression of many catabolite genes. Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as IPTG or certain sugars. LacI if the regulator protein for the lactose operon in ''E.coli'' and the hyper-spank protein of ''B. subtilis''#1(<bbpart>BBaK143015</bbpart>) and is responsible for ensuring that in the absence of lactose (or IPTG) that there is no expression trough these promoter. LacI is not endogenous to ''B. subtilis'', so LacI will need to be expressed in the host in order for the hyper-spank promoter to be regulated. In the presence of IPTG or lactose, the LacI tetramer is unable to bind DNA and so transcription resumes. This version of LacI lacks a Lva degradation tag and has a small(3 amino acid) N-terminal deletion relative to the current registry LacI (<bbpart>BBa_C0012</bbpart)> and is derivatives. The N-terminal deletion appears to be common to most of the LacI genes used in conjunction with ''B. subtilis'' though both forms are found in ''E.coli'' (in differing strains). LacI was used in conjunction with the '''Hyper-spank promoter''' (<bbpart>BBa_K143015<bbpart>) and acted as an input adaptor for a '''Polymerases per second''' (POPS) output ====References==== <biblio> #1 pmid=16166525 </biblio>
LacI was located in the sequence of the ''B. subtilis'' shuttle vector pDR111. This version of LacI lacks a Ltva degradation sequence and has a small N-terminal deletion that is observed in many LacI used in studies on ''B.subtilis''. In particular, this LacI protein is used in pDR111 to regulate expression of the inducible Phyper-spank protein (<bbpart>BBa_K143015</bbpart>) (also used in the pDR111 vector). The BioBrick prefix and suffix were applied to the gene
The LacI gene was cloned from''B. subtilis'' shuttle vector pDR111 using Pfu DNA polymerase PCR
This sequence codes for the LsrR gene which is found in the E. coli strain MG1655. When this protein fors a complex with phosphorylated AI-2 it promotes the LsrA promoter.
The DNA for this part is found in the E. coli strain MG1655.
MC31-rbs-tetR-Ter-PtetO-rbs-rfp-Ter-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter It is a test of our suicide circuit for microcystin.
MC31, tetR, PtetO, rfp, Plac, Pcons2, lacI, Ter
The 434 cI repressor protein coding sequence is a 710 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the 434 regulatory sequence, BBa_R0052. The sequence contains a LVA tag for faster degredation and has no RBS.
References (unparsed) here: Nikolnikov,S., Posfai,G. and Sain,B. The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. Gene 30 (1-3), 261-265 (1984) References (unparsed) here: Nikolnikov,S., Posfai,G. and Sain,B. The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. Gene 30 (1-3), 261-265 (1984)
Plasmid1 in detector: Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter-Plac-rbs-rfp-Ter It is a high level detection plasmid. It produces rfp when AHL is not so much.
Pcons2,luxR, Ter, Prlux, lacI, Ter, Plac, rfp, Ter
Removed LVA tag from BBa_C0052 via PCR. In pSB1AK3 currently.
Tetracycline Repressor with Consitutive Promoter BBa_J23109
The source of this part is based on the registry distribution
mnt repressor (weak) from Salmonella phage P22 (+LVA)
dimeric repressor from enterobacteriophage p22 with dissociation constant of 3*10^-8. sequence was translated with the E.Coli K12 codon usage table, and nucleotides 95-97 was changed from AAT to AAC to avoid the cut site GAATTC (94-99). the mutation R10->K10 (ctg->aaa) decreases the binding affinity of the mnt protein to its operator by approximately 12,000 fold (Kd: 2.5e-12 to 3e-8)
The ferric uptake repressor of Bacillus subtilis called YqkL is a regulator that when in contact with iron will bind and repress a regulatory sequence (the Fur box).
We sequenced the Fur box. This can be added in front of genes to provide regulation when in contact with iron.
Tetracycline Repressor with Consitutive Promoter BBa_J23116
The tetR-HNS2-90 protein fuses the polymerization demain of histone-like nucleoid-structuring (H-NS) protein to tetR. Thus, in E.coli, when TetR-HNS2-90 binds the TetR-specific DNA motif, it will recruit native H-NS to bind to adjacent DNA, which may lead to local gene silencing. This fusion protein is driven by constitutive promoter family members (BBa-J23103, BBa-J23109, BBa-J23116, and BBa-J23106). Experimental data indicates that TetR-HNS2-90 is more effective than TetR on transcription repression.
NsrR - NO(x) sensitive pYear, pNirk repressor protein
This part contains the NsrR (NO(x) sensitive pYear, pNirk repressor protein) encoding region. Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. PyeaR is regulated by the NsrR repressor protein. Nitric oxide binds to the NsrR protein and relieves the repression on PyeaR. As a result, any genes that are downstream of PyeaR are expressed. This part is also related with BBa_K2056002, the promoter region of the NirK protein, because NsrR activates the transcription of NirK under high nitrite conditions. Due to these properties, we can use this part as a sensor for oxides of nitrogen.
Because the part Bba_K1682011 was not available from the registry, we had to get it synthesized. We had to codon optimized this part to conform to assembly standards and the requirements for synthesis.
This part has been designed after the part Bba_K1682011 in the Registry of Standard Biological Parts.
tetracycline repressor from transposon Tn10 (+LVA)
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). Lutz R, Bujard H., Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). Lutz R, Bujard H., Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 BBa_C0040 TetR Protein is based on the TetR sequence from Elowitz's repressilator. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
Temperature Sensitive Lamda cI Repressor
The temperature sensitive lambda cI protein has the cI857 mutation. Therefore, the repressor is denatured at 42??C. It normally represses promoter activity from bacteriophage lambda PL and PR promoters, and has maximal activity at the permissive temperature of 30??C. cI1 However, when the temperature is raised further, typically 42??C, the functionality of such protein is lost and it is no longer able to bind the operator of the promoter. cI2
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
A composite part with coding sequence for LacI-LVA together with ribosome binding site (RBS) and a double transcriptional terminator. Fore use in regulation of LacI-repressed genetic elements, by combining the part with an appropriate promoter.
This part is a reconstruction of BBa_K292006. BBa_K292006 has the same theoretical sequence, but the DNA available through the registry does not match this sequence. We thus offer this part as a replacement.
Tetracycline Repressor with Consitutive Promoter BBa_J23103
The SsrA at the end of BBa_C0040 is not present in our design.
The source of this part are all from the registry distributions.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
represillator of Elowitz and Leibler (2000)
This is a TetR protein generator regulated by the constitutive promoter J23111. The protein has a LVA tag for degradation.
Pcons2-rbs-luxR-Ter. It achieves a constant expression of luxR, the AHL combining protein Pcons2 is BBa_J23100
The marR gene encodes a repressor of the marRAB operon
Amplified from MG1655 (Accession NC_000913) chromosome using primers listed below marR_BB_F TTCGAATTCGCGGCCGCTTCTAGATGAAAAGTACCAGCGATCT marR_BB_R GGCCGCTACTAGTATTATTACGGCAGGACTTTCTT
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.
LacI repressor (temperature-sensitive mut 265) (+LVA)
Mutated from BBa_C0012 Repressor, LacI (LVA+) to be temperature sensitive. Mutation from Chao, et al., 2002: Gly265->Asp (GG(803)T->GAT)
Pcons2-rbs-lacI-Ter. It constantly produces lacI.
lacI IS mutant (IPTG unresponsive) R197A
Short description: The lacI IS mutant is almost identical to the lacI transcriptional regulator except for the difference that it is not able to bind IPTG or allolactose due to a mutation; it therefore can not be activated by induction with these substances. Since it recognizes the same motif in the lac promotor region, it strongly represses transcription of all genes regulated by promotors with lacI binding site even if IPTG or allolactose are present. It can be used to terminate the expression of proteins under lac control if IPTG can not be removed from the cell rapidly. Detailed description: Expression of the lac operon in E. coli is tightly controlled by lacI, a protein, which binds to a repressor binding site within the promotor and disables transcription by obscuring the promotor region. When bound to DNA, lacI is in the tetrameric form, which consists of two dimers interacting at the end distal from the DNA binding site. Upon binding of allolactose or IPTG, the tetramer breaks down into two dimers and the affinity for the repressor binding site is greatly reduced; the lacI IPTG complex will diffuse away from the repressor binding site, leaving the promotor accessible. As a result of decades of genetic and structural studies, the function of lacI is now understood on the molecular level (1, 2). Mutational experiments have identified residues, which abolish IPTG response upon mutation (3). Furthermore, the x-ray crystal structure of lacI with bound IPTG has allowed the identification of residues that interact with IPTG and which are promising targets for mutagenesis (1). We decided to mutate residues R197 and T276, which are located in the IPTG binding groove, contact IPTG and have been shown to produce the lacI IS mutation in previous genetic experiments. Since a quantitative study of the strength of inhibition by different lacI IS mutants has to our knowledge not been published so far, we decided to generate a set of eight mutated lacIs, in which we replaced either R197 with alanine or phenylalanine or T276 with alanine or phenylalanine or both in all possible combinations. lacIIS-1: R197A lacIIS-2: R197F lacIIS-3: T276A laciIS-4: T276F lacIIS-5: R197A T276A lacIIS-6: R197A T276F lacIIS-7: R197F T276A lacIIS-8: R197F T276F
During ste-directed mutagenesis, the codons to be mutated were replaced with the most highly utilized codons in E. coli to prevent complications from the use of rare codons. The lacI IS sequences were analyzed for BioBrick restriction sites within the coding sequence to ensure their compatibility.
Source: The lacI IS mutants presented derive from the BioBrick holding lacI by itself (C0012). Site-directed mutagenesis was performed by PCR, subsequent DpnI digest and transformation. The following primers were used: R197F forward CGGCGCGTCTGTTTCTGGCTGGCTG R197A forward CGGCGCGTCTGGCGCTGGCTGGCTG R197F reverse CAGCCAGCCAGAAACAGACGCGCCG R197A reverse CAGCCAGCCAGCGCCAGACGCGCCG T276F forward GGATACGACGATTTTGAAGACAGCTC T276A forward GGATACGACGATGCGGAAGACAGCTC T276F reverse GAGCTGTCTTCAAAATCGTCGTATCC T276A reverse GAGCTGTCTTCCGCATCGTCGTATCC All lacI IS holding BioBricks were verified by sequencing.
lacI IS mutant (IPTG unresponsive) T276A
PgolTS-golS-PgolB-tetR-Ter-PtetO-rfp-Ter. If there exist gold ions, rfp will not be produced.
PgolTS-golS-PgolB, tetR, PtetO, rfp, Ter
lacI IS mutant (IPTG unresponsive) R197F T276A
Coding for the trans-acting regulator SmtB
In 1993, Huckle and co-workers reported the isolation and analysis of the smt locus from Synechococcus sp.. Gene smtB, one of the components of the locus, encodes a transcriptional repressor of gene smtA, with which is required for normal tolerance to Zn2+ and Cd2+. The product of smtB functions by binding to the smt operator-promoter(smtO-P) and dissociating in the presence of Zn2+ due to the classical helix-turn-helix motif similar to many DNA-binding proteins. It can also be genomically rearranged when induced by Cd2+, leading to a deletion within smtB because of a highly iterated palindrome (HIP1), a highly represented octanucleotides(5′-GCGATC-GC-3′) which traverse both borders of the excised element. These two mechanisms both cause the elevated expression of smtA.
According to the mechanisms of smt locus represented above, gene smtB should function with the accompany of smt operator-promoter. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.
Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals. In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium Synechococcus sp.. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like. Owing to the works of Robinson and Huckle, the corresponding locus of Synechococcus PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.
NsrR protein is a nitrate sensitive transcriptional repressor. When there is nitrate, it will be oxidized to nitric oxide by nitrate oxidase, which in turns relieve the repression of NsrR on PyeaR promoter.
lacI IS mutant (IPTG unresponsive) R197F
PcI-rbs-lacI-Ter. It produces enough lacI when there is less cI.
cI repressor from E. coli phage lambda (+LVA)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator (BBa_R0051).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 BBa_C0051 cI repressor is based on the cI repressor from the Elowitz's repressilator. It has been modified to include a rapid degradation LAA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
This is a TetR protein generator regulated by the constitutive promoter J23109. The protein has a LVA tag for degradation.
lacI IS mutant (IPTG unresponsive) R197A T276A
Human phosphodiesterase 5A gene silencing device NO.2
This device is uesd for silencing the human phosphodiesterase 5A (PDE5A) gene.A U6 promoter driving a designed, synthetic shRNA-like miRNA followed by the terminator. PDE5A is a cGMP-binding, cGMP-specific phosphodiesterase, a member of the cyclic nucleotide phosphodiesterase family. This phosphodiesterase specifically hydrolyzes cGMP to 5'-GMP. It is involved in the regulation of intracellular concentrations of cyclic nucleotides and is important for smooth muscle relaxation in the cardiovascular system. We designed 3 silencing device and test their function at the same time.Here is the another two device :BBa_K1720003,BBaK172005 This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa to detect cGMP level. The results are as follow:
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.
global DNA-binding transcriptional dual regulator
PCR using primers with biobrick prefix and surfix, and then cloned into pSB1AC3.
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>
This is a variation of the LacI protein that acts as a repressor for the pLac promoter and is repressed by IPTG. It has a point mutation at the third codon where proline is replaced by tyrosine. This missense mutation causes an increased binding affinity of the LacI_I12 protein with the pLac promoter.
Because there are many codons that code for the amino acid tyrosine, we chose to use the TAT codon, because it was the most common tyrosine codon found throughout the rest of the LacI wild-type gene sequence.
The basic sequence comes from Ecogene.com
MC7-rbs-tetR-Ter-PtetO-rbs-rfp-Ter-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter It is a test of our suicide circuit for microcystin.
MC17, tetR, PtetO, rfp, Plac, Pcons2, lacI, Ter
lexA3 is lexA (BBa_K864201) with a G85D mutation (G>A basepair change at position 254)
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)
Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter It produces luxI when there is enough IPTG.
This is a TetR protein generator regulated by the constitutive promoter J23106. The protein has a LVA tag for degradation.
lacI IS mutant (IPTG unresponsive) R197F T276F
LacI wild-type gene sequence. Repressed by IPTG
We changed the start codon from CTG to ATG
The gene sequence was obtained from Ecogene.com
rox1 is a repressor. It targets the promotor anb1.
Sequence and information is based on the Saccharomyces Genome Database, see http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=rox1