pTet promoter + Lock (crRNA) + ccdB + dterm
The components for this part were obtained from the iGEM Registry of Standard Biological Parts.
pTet is a constitutive promoter, meaning that the Cis+ccdB gene is continuously transcribed; however, because the Cis portion of the mRNA forms a hairpin which blocks the RBS, the gene cannot be translated. In this way, the Cis acts as a "lock" which will only allow translation, and therefore cell death, once opened. We designed a separate gene to act as a "key," controlled by a UV-light-inducible promoter (BBa_K1300000).
We took the pTet+Cis out of the Registry and added a toxin behind it in order to test the riboregulatory system, by means of a kill switch.
Double terminator consisting of BBa_B0010 and BBa_B0012
The ccdB gene was obtained from the U-GENT vector pK7FWG2.0
The ccdB gene is a topoisomerase II poison and will kill most cell lines. This is an improvement to the prior ccdB cell death gene (BBa_P1010) in that the size has been reduced and it is now easier to use. CcdB can be used as a selectable marker by mixing it with the desired structure and a plasmid cut with BioBrick restriction enzymes. After ligation and transformation, because ccdB is toxic to most cell lines, the only visible colonies should contain the desired structure in the desired plasmid. CcdB is toxic to most cell lines with the exception of DB3.1 cell lines (BBa_V1005).
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.
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
Biobricked version of Isaacs' riboregulator cis repressed lock, crR12.
Conversion of the iGEM parts registry to SBOL2.1