DNAseI generator inhibited by recombinant pCat promoter
DNAseI generating module that is inhibited by (lack of) recombination. Initially, the promoter is set to produce actin with a fast degradation tag, which inhibits DNAseI. When fimE is expressed elsewhere in the cell, the promoter is flipped to stop production of actin. Instead, DNAseI is expressed, along with ECP32 which degrades actin. Additionally, tetR is produced to be used in other regulatory mechanisms.
There is a terminator before the pCat promoter to prevent "backwards" expression, which would be detrimental to the strong "digital" behavior the recombinant region attempts to achieve. ** Everything before the fimE IRL should be on the antisense strand, and thus inverted.
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.
fimE inverted repeat left recombination site
Constitutive Promoter, Medium Transcription
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
AANDENYALAA. (Very fast) SsrA degradation tag.
C-terminal degradation tags are commonly found in high turnover proteins in Escherichia coli.
This sequence codes for the amino acid sequence AANDENYALAA, which when fused to the C-terminal of proteins, will make the protein susceptible to very fast degradation through SspB-mediated binding to the ClpX protease. The following rates of degradation of this tag are pulled from the corresponding references below: ~5 Vmax/ [Clpx6] min-1 from (1) ~0.5%/min on log scale from (2) ~1 min half life from (3) See the following references for further information on degradation rates and mechanisms of this tag: (1)McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701. (2)Flynn et al 2003. Mol. Cell. 11: 671. Flynn et al. 2001. PNAS 98(19): 10584. Anderson et al 1998. App. Env. Microbiol. 64(6):2240.
C-terminal tag. Degradation rate is very fast. Deviations from this sequence in key amino acids will lower degradation rates (see Parts BBa_M0051, BBa_M0052, BBa_M0053). Three C-terminal aa's (LAA in this case) are necessary and sufficient for ClpX binding and degradation. Upstream aa sequence serves as a binding site for SspB, which guides rapid binding to ClpX.
fimE inverted repeat right recombination site
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.
Conversion of the iGEM parts registry to SBOL2.1