Part:BBa_K4817009
IPTG-induced kill switch
Our quorum sensing suicide system was induced and regulated using IPTG in the prophase and metaphase of characterization validation, Part1 composed of LacO/LacI (BBa_K1624002, BBa_K3257045), pT7 (BBa_K4609008) and ccdB ( BBa_P1010 ) compositions, we constructed part1 aiming to verify the expression of CcdB toxic protein in the chassis and its characterization of bactericidal function through IPTG induction experiments. CcdB is a toxic protein in the CcdB/CcdA toxin-antitoxin system, with a half-life of more than 2h after expression, which, as an intracellular toxin, induces the formation of the unresolvable covalent by disrupting DNA deconjugating enzyme activity mainly through GyrA-DNA complex. CcdB promotes the breakage of chassis plasmid and chromosomal DNA, ultimately leading to defective chassis division and killing the chassis [1]. LacO/LacI is commonly found in the pET series of plasmids, and IPTG (Isopropyl β-D-1-Thio-galactopyranoside), a molecular analog of iso-lactose, which enters into the intracellular system as a low-toxicity exogenous substance, is not able to be metabolized by common laboratory chassis, such as Escherichia coli. IPTG is a molecular analog of iso-lactose. IPTG functions in the same way as isolactose in that it acts as an inducer in the chassis to bind to the deterrent factor in the Lac operon, which in turn prevents LacI from binding to LacO, ultimately initiating the expression of the target genes downstream of pT7; when there is no IPTG, LacI binds to LacO to deter the expression of CcdB [2].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1813
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1263
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