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 will be constructed by cloning Laciq-T7ccdB with standard Biobrick prefix and suffix in pSB1C3.
Journal
July
Week 3
Introduction given by our lab instructors
General techniques: plasmid/PCR purification, inoculation, gel extraction, restriction & ligation.
Safety and waste disposal training
Introduction to CloneManager
General preparations: sterile mQ, sterile eppendorf
August
Week 1
Experiment 1
Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- 362.4 ng/µL
Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp
Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL
HiFi PCR using Primestar polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589. Analytical gel: negative
PCR using Q5 polymerase on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589. Analytical gel: negative
"
