Week 6

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  • Miniprep for P0412 and R0010.
  • We inoculate a colony of J04431 in 5 ml 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 5 ml in 2 tubes;

-fluorescence control: bacterial cells from one tube are fluorescent and cells from the other not;

-add glucose 2 mM in each falcon;

-incubation at 37°C for 1 h;

-there are no fluorescence bacteria in the two cultures;

-after 2 hours 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 plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we resuspend in 5 ml of LB. Bacteria become fluorescent. When we add 2 mM of glucose the bacteria are no fluorescent.

So, it is possible to control Plac activation with glucose. As it's known in literature, endogenous LacI is not enough to repress Plac promoter from a high copy number plasmid since a small amount of glucose arises the cinetic rateo of Plac trascription. We are going to insert LacI in our construct to solve this problem. This test enables us to verify GFP emivita which is about 40 minutes (link literature).

  • Model analysis.