Part:BBa_K191005 for TrpR-RBS-TetR-Term-pTetR (PCR to isolate this segment) Part:BBa_B0034 for sequence of RBS (added RBS by PCR to CI, AraC, and Mnt)
This part is a double repressor system derived from Part:BBa_K191005. A Trp promotor controls expression of tetR, a repressor to the Tet promotor. The Trp promotor is ON, unless otherwise repressed. The expression of CI, AraC, and Mnt, which follow the Tet promotor, is inhibited given the absence of tryptophan. ==Usage== This part is designed to interact with Part:BBa_K191003, as the light-activated LovTAP binds to and represses the Trp promotor. Thus, given additional expression of LovTAP, the presence of light causes the repression of TetR expression and the activation of expression of CI, AraC, and Mnt. You can see the full circuit design below - press the light button to toggle the circuit..
Because there are illegal SpeI sites in the Trp promotor, all assembly had to be done with RFC 21 standard (bgl bricks). Thus, we had to add the appropriate bgl-brick sites to all parts we worked with. See Team:Brown/Project/LRC_overview for a more in depth description.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
cI repressor from E. coli phage lambda (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
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.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
tetracycline repressor from transposon Tn10 (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
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.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
araC arabinose operon regulatory protein (repressor/activator) from E. coli (+LVA)
GenBank: NC_002655 (www.ncbi.nlm.nih.gov)
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)
Sequence of Trp operon was found at : http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00067 Part was added to the system as the upstream sequence of the forward primer in a PCR.
Promoter of Tryptophan operon ( Tryptophan operon is an operon that codes for the components of tryptophan's synthesis)
2 SpeI enzyme restriction sites on Trp operon sequence.
Double terminator consisting of BBa_B0010 and BBa_B0012
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
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.
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
Conversion of the iGEM parts registry to SBOL2.1