- I763005 digestion with Spe1/Pst1;
- Ligation for J04500+J22101 (I763012);
- We inoculate a P0412, R0010 colony in 5ml.
- Miniprep for P0412 and R0010.
- We inoculate a colony of J04431 in 5ml to perform the fluorescence test with glucose (see the following protocol).
J04431 bacteria from glycerol stock:
-growth of 10 aliquots (1 ml/each) at 37°C for 1h;
-collection of 5ml in 2 tubes;
-fluorescence control: bacterial cells from one tube are fluorescent and cells from the other are not;
-add glucose 2mM in each falcon;
-incubation at 37°C for 1h;
-there are no fluorescent bacteria in the two cultures;
-after 2h the fluorescence is the same.
We check bacteria from the stock and we see that some of them are fluorescent, even if we don’t expect this result.
- We strake on plates from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we re-suspend in 5ml of LB. Bacteria become fluorescent. When we add 2mM of glucose bacteria are not fluorescent.
So, it is possible to control PLac activation with glucose. As it is known from literature, endogenous LacI is not sufficient to repress the PLac promoter in a high copy number plasmid since a small amount of glucose increases the kinetic ratio of PLac transcription. We are going to insert LacI in our construct to solve this problem. This test enables us to verify GFP half-life which is about 40 minutes.
- J04431 digestion with Eco/Xba;
- B0015 digestion with Eco/Xba;
- P0412 digestion with Xba/Pst1;
- J04431, B0015, P0412 band extraction;
- Transformation of J04500+J22101 (I763012) ligation.
- Model analysis.
- Miniprep of I763012 and of I763016;
- I763012 digestion with Spe1/Pst1;
- I763012 control digestion with Xba/Pst1;
- I763012 extraction from gel.