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 1 ml 10 aliquot at 37°C for 1h;

-collection of 5 ml in 2 falcon;

-fluorescence control (1 green, 1 not);

-add glucose 2 mM in each falcon;

-incubation at 37°C for 1 h;

-in each falcon there aren't fluorescence bacteria;

-idem after 2 hours.

Perhaps we have problem with the stock, infact we see some fluorescent bacteria in 10 ul of the stock on the micro slide, 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 risuspend in 5 ml of LB. We observe the bacteria become fluorescent. When we add 2 mM of glucose the bacteria don’t become fluorescent.

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

  • Model analysis.