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 in 5 ml for P0412, R0010.
+
*We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].
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::'''08/07/07'''
::'''08/07/07'''
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*Miniprep for P0412 and R0010.
+
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].
-
*We inoculate a colony of  J04431 in 5 ml to perform the fluorescence test with glucosio (see the following protocol).
+
*We inoculate a colony of  [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucosio (see the following protocol).
-
'''J04431 bacteria from glycerol stock:'''
+
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''
''-grew of 10 aliquote da 1 ml at 37°C for 1h;''
''-grew of 10 aliquote da 1 ml at 37°C for 1h;''
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We are going to insert in our costruct esogen LacI to resolve this problem.
We are going to insert in our costruct esogen LacI to resolve this problem.
This test enable us to verify GFP emivita which is about 40 minutes as is know in literature.
This test enable us to verify GFP emivita which is about 40 minutes as is know in literature.
-
*J04431 digestion with Eco/Xba;
+
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;
-
*B0015 digestion with Eco/Xba;
+
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;
-
*P0412 digestion with Xba/Pst1;
+
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;
-
*J04431, B0015, P0412 band extraction;
+
*[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.  
*Transformation of  [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation.  
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::'''08/09/07'''
::'''08/09/07'''
-
*R0051+P0412 (I763010) ligation;
+
*[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.
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.
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::'''08/10/07'''
::'''08/10/07'''
-
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of I763016;  
+
*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] digestion with Spe1/Pst1;
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;

Revision as of 11:48, 28 August 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 glucosio (see the following protocol).

[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:

-grew of 10 aliquote da 1 ml 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 controll the Plac attivation with glucose. As is know in literature endogene LacI is not sufficient to repress it and a little quntity of glucose arises the cinetic rateo of Plac trascription. We are going to insert in our costruct esogen LacI to resolve this problem. This test enable us to verify GFP emivita which is about 40 minutes as is know in 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] estraction from gel.




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