Week 6

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(Difference between revisions)
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''-idem after 2 hours.''
''-idem after 2 hours.''
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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 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 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.
+
*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.
-
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.
+
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 and 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).
This test enables us to verify GFP emivita which is about 40 minutes (link literature).
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;

Revision as of 13:52, 3 September 2007

08/06/07
  • [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;
  • Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]);
  • We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].



08/07/07
  • Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].
  • We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucose (see the following protocol).

[http://partsregistry.org/Part:BBa_J04431 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.

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 and 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).

  • [http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;
  • [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;
  • [http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;
  • [http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;
  • Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation.



08/08/07
  • Model analysis.


08/09/07
  • [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;
  • We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.



08/10/07
  • Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016];
  • [http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;
  • [http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;
  • [http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel.




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