Week 6

From 2007.igem.org

(Difference between revisions)
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* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;
* [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]);  
*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 a [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010]colony in 5ml.
+
*We inoculate a [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010] colony in 5ml.
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::'''08/07/07'''
::'''08/07/07'''
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].
*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).
+
*We inoculate a colony of  [http://partsregistry.org/Part:BBa_J04431 J04431] in 5ml to perform the fluorescence test with glucose (see the following protocol).
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''
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''-collection of 5ml in 2 tubes;''
''-collection of 5ml in 2 tubes;''
-
''-fluorescence control: bacterial cells from one tube are fluorescent and cells from the other not;''
+
''-fluorescence control: bacterial cells from one tube are fluorescent and cells from the other are not;''
''-add glucose 2mM in each falcon;''
''-add glucose 2mM in each falcon;''
Line 23: Line 23:
''-incubation at 37°C for 1h;''
''-incubation at 37°C for 1h;''
-
''-there are no fluorescence bacteria in the two cultures;''
+
''-there are no fluorescent bacteria in the two cultures;''
''-after 2h the fluorescence is the same.''
''-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 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 re-suspend in 5ml of LB. Bacteria become fluorescent. When  we add 2mM of glucose the bacteria are no fluorescent.
+
*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'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 kinetic ratio of PLac transcription.
+
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.
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 half-life 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;
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;

Revision as of 10:28, 5 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 a [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010] colony in 5ml.



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 5ml to perform the fluorescence test with glucose (see the following protocol).

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