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Team:Paris Saclay/Notebook/August/30
From 2013.igem.org
(→6 - Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3 in DH5α) |
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We incubate it at 37°C with Xgal in anaerobic conditions for NarK with RBS-LacZ-Term in PSB3K3. We incubate it at 37°C with Xgal in aerobic conditions for NarK with Pfnr-LacZ-Term in PSB3K3. | We incubate it at 37°C with Xgal in anaerobic conditions for NarK with RBS-LacZ-Term in PSB3K3. We incubate it at 37°C with Xgal in aerobic conditions for NarK with Pfnr-LacZ-Term in PSB3K3. | ||
| + | ===='''7 - Electrophoresis of Colony PCR of Pfnr with RBS_AmilCP-Term in DH5α '''==== | ||
| + | |||
| + | XiaoJing | ||
| + | |||
| + | {| | ||
| + | | style="width:350px;border:1px solid black;" | [[]] | ||
| + | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
| + | * Well 1 : 6µL DNA Ladder | ||
| + | * Well 2 : 20µL of Pfnr with RBS_AmilCP-Term clone 1+4µL of 6X loading dye | ||
| + | * Well 3 : 20µL of Pfnr with RBS_AmilCP-Term clone 2+4µL of 6X loading dye | ||
| + | * Well 4 : 20µL of Pfnr with RBS_AmilCP-Term clone 3+4µL of 6X loading dye | ||
| + | * Well 5 : 20µL of Pfnr with RBS_AmilCP-Term clone 4+4µL of 6X loading dye | ||
| + | * Gel : 1.0% | ||
| + | |} | ||
| + | |||
| + | Expected sizes : | ||
| + | * Pfnr with RBS_AmilCP-Term : 1000bp | ||
| + | |||
| + | {| | ||
| + | | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" | | ||
| + | We obtain fragments at the right size. | ||
| + | |} | ||
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''=== | ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''=== | ||
Revision as of 14:07, 2 October 2013
Notebook : August 30
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Results of liquid culture of Pfnr-RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions
XiaoJing
|
IT WORKS !!! |
In anaerobic conditions, thank to promotor Pfnr, RBS-Amil CP-Term gene is activated and we can see a violet coloration of bacterias.
In aerobic conditions, promotor Pfnr is inactivated, RBS-Amil CP-Term isn't activated and we can't see any coloration of bacterias.
2 - Results of culture of Pfnr with RBS-Amil CP-Term in PSB1C3, NirB with RBS-LacZ-Term in PSB1C3 in aerobic and anaerobic conditions
XiaoJing
|
Purification of 08/29/13 didn't work. We have blue colonies for Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for NirB with RBS-LacZ-Term in PSB1C3 in anaerobic conditions. |
3 - Ligation of Pfnr with RBS-LacZ-Term in PSB1C3
XiaoJing
Used quantities :
- Pfnr : 8µL
- RBS-LacZ-Term in PSB1C3 : 3µL
- Buffer ligation : 2µL
- Ligase : 1µL
- H2O : 6µL
4 - Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB1C3 in DH5α
XiaoJing
Protocol : Bacterial transformation
We incubate it at 37°C with Xgal in aerobic conditions.
5 - Ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3
XiaoJing
Used quantities :
- Pfnr, NarK : 3µL
- RBS-LacZ-Term : 3µL
- PSB3K3 : 5µL
- Buffer ligase : 2µL
- Ligase : 1µL
- H2O : 6µL
6 - Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3 in DH5α
XiaoJing
Protocol : Bacterial transformation
We incubate it at 37°C with Xgal in anaerobic conditions for NarK with RBS-LacZ-Term in PSB3K3. We incubate it at 37°C with Xgal in aerobic conditions for NarK with Pfnr-LacZ-Term in PSB3K3.
7 - Electrophoresis of Colony PCR of Pfnr with RBS_AmilCP-Term in DH5α
XiaoJing
| [[]] |
|
Expected sizes :
- Pfnr with RBS_AmilCP-Term : 1000bp
|
We obtain fragments at the right size. |
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins
1 - Electrophoresis of Colony PCR of FNR, RBS-FNR and RBS-BphR2
| ]] |
|
Expect sizes :
- FNR : 1096 bp
- RBS-FNR : 1014 bp
- RBS-BphR2 : 1469 bp
|
We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good. |
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