pCon from BBa_J23119, RBS from BBa_B0034, tetR from BBa_C0040, DT from BBa_B0015,PTet from BBa_R0040, pT7 from BBa_K525998, GFP from BBa_E0040. First terminator comes from BBa_B0013, while the second terminator from BBa_B0015. T7RNAP is PCR amplified from genome of BL21.
This part is a complete regulatory device sensitive to tetracycline. The first promoter is constitutively on and can express tetR, a protein that can repress the downstream promoter pTet in the absence of tetracycline(Tc). As a result, no T7RNAP is produced, resulting in the inactivity of pT7. Thus, no GFP is expressed and no fluorescence can be detected. In the presence of Tc, however, tetR can be repressed by Tc and repression over pTet can be removed, resulting in the expression of T7RNAP and activation of pT7 which further leads to the expression and amplification of GFP. Under such circumstances, positive feedback can be achieved and obvious fluorescence can be observed. This part is used to test the tet switch and the tet-on/tet-off system.
This part is used for logic verification. Whether green fluorescence is observed serves as an indicator whether the system works. In real use, substitute GFP for target gene.
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.
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
Double terminator consisting of BBa_B0010 and BBa_B0012
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.
Overlap extension of synthetic oligonucleotides
Bacteriophage T7
Gene 1 coding region from bacteriophage T7 encoding a RNAP polymerase (RNAP). This single-protein RNAP recognizes a 21 bp consensus promoter sequence and is orthogonal to E. coli RNAP.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Strong transcriptional terminator consisting of a 20 bp stem-loop that has been engineered to be bidirectional.
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). Subsequently mutations were introduced to make the terminator bi-directional (i.e., AAAAAA insertion on 5' side of the stem loop). Additional mutations were introduced on the 3' side of the stem loop to increase the number of T's and eliminate any promoter -10 site that might be present to avoid initiation of transcription of whatever is downstream. Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
This part is the T7 promoter without RBS. It has been reported that T7 promoter doesn't work with E.coli RNA polymerase, but only with T7 RNA polymerase(T7RNAP). However, we observed leakage of it in the absence of T7RNAP.
We deleted the RBS in BBa_K525998, so that RBS with different efficiency can be used.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1