P(Lac)IQ RBS Aiia RBS LuxR ter ter luxpR RBS tetR ter ter
We designed and built a kill switch mechanism that is based on the activation of a lethal gene downstream of an inducible promoter that is activated over time. The presented part represents the first and the second module of our designed genetic circuit. The here presented BioBrick consists of a constitutive promoter (I14032) driving expression of Aiia and LuxR followed by a Lux promoter (R0062) , driving expression of TetR. By using this part one can control the amount of producing TetR which means that at the beginning the system will produce TetR. After a certain amount of time thatdepends on the initial concentration of AHL , the system will stop producing TetR due to the absence of AHL that interacts with LuxR to form its active conformation. The scheme below explains how this BioBrick works:
P(Lac)IQ RBS Aiia RBS LuxR term term luxpR
parts registry and gBlocks ordered from idt
We designed and built a kill switch mechanism that is based on the activation of a lethal gene downstream of an inducible promoter that is activated over time. The presented part is the first module of our designed genetic circuit (BBa_K1767003). In order to characterize and understand each module of the genetic circuit we designed two composite parts that allow us to verify the (1) the degradation rate of AHL by AiiA and (2) induction of the pLux promoter by LuxR in presence of AHL with and without co-expression of AiiA. (1) The here presented BioBrick consists of a constitutive promoter driving expression of AiiA and LuxR followed by a Lux promoter which drives expression of a fluorescent reporter (here: yellow fluorescent protein). For a schematic overview see the figure below
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.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
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.
Conversion of the iGEM parts registry to SBOL2.1