Week 6
From 2007.igem.org
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::'''08/07/07''' | ::'''08/07/07''' | ||
*Miniprep for P0412 and R0010. | *Miniprep for P0412 and R0010. | ||
| - | *We inoculate a colony of J04431 in 5 ml to perform the fluorescence test with glucosio (see. 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 inoculate a colony of J04431 in 5 ml to perform the fluorescence test with glucosio (see the following protocol). |
| + | |||
| + | ''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. | *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. | 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. | ||
Revision as of 09:41, 28 August 2007
- 08/06/07
- I763005 digestion with Spe1/Pst1;
- Ligation for J04500+J22101 (I763012);
- We inoculate in 5 ml for P0412, R0010.
- 08/07/07
- Miniprep for P0412 and R0010.
- We inoculate a colony of J04431 in 5 ml to perform the fluorescence test with glucosio (see the following protocol).
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.
- J04431 digestion with Eco/Xba;
- B0015 digestion with Eco/Xba;
- P0412 digestion with Xba/Pst1;
- J04431, B0015, P0412 band extraction;
- Transformation of J04500+J22101 (I763012) ligation.
- 08/08/07
- Model analysis.
- 08/09/07
- R0051+P0412 (I763010) ligation;
- We inoculate J04500+J22101 (I763012) ligations.
- 08/10/07
- Miniprep of I763012 and of I763016;
- I763012 digestion with Spe1/Pst1;
- I763012 control digestion with Xba/Pst1;
- I763012 estraction from gel.
