Notebook > Assembly > Assembling (three plasmids strategy)
Assembling (three plasmids strategy)
Resource
Bistable on pSB1A2 and pSB6A1 form me;
AND gate: the best AND gate us had constructed (pBad-SupD, pSal-T7ptag, RBS B0033);
CI on pSB3T3: from Haoqian Zhang
2009.10.4
Transformation: put bistable into E.coli
Plasmids: Bistable on pSB1A2 and pSB6A1,
Plate on Amp LB plates.
2009.10.5
Prepare competent cell
E.coli contained bistable plasmid,
Transformation: put AND gate into E.coli (contained bistable)
Plasmid: AND gate,
Plate on Amp & Kan LB plates;
2009.10.6
Prepare competent cell
E.coli contained bistable & AND gate plasmids,
Transformation: put AND gate into E.coli (contained bistable & AND gate)
Plasmids: six different T7-CI,
Plate on Amp, Kan & Tet LB plates;
2009.10.7-2009.10.10
Inducing test
Then the colonies contained three plasmids were picked into 3 mL liquid LB and incubated until the OD600 value reached 0.4~0.6. At this time, we used different combinations of different concentration of arabinose and salicylate to induce. Every two hours we discarded 2mL LB culture, and added 2mL new LB into it, and the concentration of inducers should be kept. Repeating this step for 3 times, and then cultivated the culture overnight to ensure saturated induction and interaction between different modules. The results were tested by flowcytometry.
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