To test our idea we are conducting 6 experiments. These are described below.
Experiment 1
Experiment 1 is the knock-in (KI) of the construct containing ccdA, GFP and the homologous regions (this was constructed beforehand). This will be done by using the method of Datsenko & Wanner [PNAS 2000], based on homologous recombination. The principle of the KO/KI method is depicted below:
Datsenko-Wanner, 2000
Experiment 2
In experiment 2 a plasmid containing ccdB under control of a T7 promotor will be constructed.
Experiment 3
The plasmids constructed in experiment 2 will now be tranformed in the E. coli strain constructed in experiment 1.
Experiment 4
Strains constructed in experiment 3 will be used to perform CIChE. Tanden gene replication of reporter protein GFP will be induced by replicating the antitoxin ccdA as a response on titration of the toxin ccdB. Titration of ccdB under inducible T7-promoter will be accomplished by different levels of IPTG [0.01 mM – 0.5mM] and different plasmid copy numbers [p5, p10 and p20].
Experiment 5
Experiment 6
In this experiment, a new part, called BBa_K1105000, will be constructed by cloning Laciq-T7ccdB with standard Biobrick prefix and suffix in pSB1C3. Laciq-T7ccdB is derived from the plasmid p10-LacIq-T7ccdB, which originally
comes from a mini F-plasmid positive strains (such as E. coli F+). The function of this part is to produce CcdB (in control of a T7 promotor), which interferes with the topoisomerase unit gyrA.
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