"
Team:Paris Saclay/Notebook/August/29
From 2013.igem.org
(Difference between revisions)
| Line 264: | Line 264: | ||
==='''A - Aerobic/Anaerobic regulation system'''=== | ==='''A - Aerobic/Anaerobic regulation system'''=== | ||
| - | ===='''Objective : characterize Bba_K1155000 and Bba_K1155004'''==== | + | ===='''Objective : characterize Bba_K1155000 and Bba_K1155004, Bba_K1155005, Bba_K1155006'''==== |
===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions'''==== | ===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions'''==== | ||
| Line 373: | Line 373: | ||
* Well 1 : 6µL DNA Ladder | * Well 1 : 6µL DNA Ladder | ||
* Well 2 : 2µL of RBS-LacZ-Term clone 10+1µL of 6X loading dye | * Well 2 : 2µL of RBS-LacZ-Term clone 10+1µL of 6X loading dye | ||
| - | * Well | + | * Well 3 : 2µL of RBS-Amil CP-Term clone 9+1µL of 6X loading dye |
* Gel : 1.0% | * Gel : 1.0% | ||
|} | |} | ||
| Line 385: | Line 385: | ||
We obtain fragments at the right size for RBS-LacZ-Term. We will ligate it. | We obtain fragments at the right size for RBS-LacZ-Term. We will ligate it. | ||
|} | |} | ||
| + | |||
| + | ===='''5 - Electrophoresis to check the digestion of Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI'''==== | ||
| + | |||
| + | 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 : 5µL of NirB clone 7+1µL of 6X loading dye | ||
| + | * Well 3 : 5µL of NarG clone 6+1µL of 6X loading dye | ||
| + | * Well 4 : 5µL of Bba_K1155006 already digested by SpeI+1µL of 6X loading dye | ||
| + | * Well 5 : 5µL of Bba_K1155004 already digested by SpeI+1µL of 6X loading dye | ||
| + | * Well 5 : 5µL of Bba_K1155005 already digested by SpeI+1µL of 6X loading dye | ||
| + | * Gel : 1.0% | ||
| + | |} | ||
| + | |||
| + | Expected sizes : | ||
| + | * NarK, NarG, NirB : ... | ||
| + | |||
| + | {| | ||
| + | | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" | | ||
| + | We obtain fragments at the right size for NarK. We will ligate it | ||
| + | |} | ||
| + | |||
((((((((((((((===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''==== | ((((((((((((((===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''==== | ||
| Line 421: | Line 446: | ||
|} | |} | ||
| - | ====''' | + | ===='''3 - Gel purification of the digestion of Bba_J04450 by PstI/SpeI'''==== |
XiaoJing | XiaoJing | ||
| Line 428: | Line 453: | ||
Nanodrop : | Nanodrop : | ||
| - | * Pfnr : 7.2ng/µL | + | * Pfnr : 7.2ng/µL))))))))))))))))))))))) |
Revision as of 00:50, 22 September 2013
Notebook : August 29
summary
- Digetion of RBS_AmilCP+Term_PSB1C3 and RBS_LacZ+Term_PSB1C3 by SpeI and PstIand check the size by Gel electrophoresis
- check the size by Gel electrophoresis for Degestion product of promoter fnr(activator)
- We do PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
- we got blue color of ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.
lab work
- A.aero/anaerobic regulation system
- Digestion for PSB3K3
| DNA | 9µl |
| Ecoil I | 2µl |
| PST I | 2µl |
| H2O | 5µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_LacZ+Term_10(08/09/13)
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_LacZ+Term_11(08/09/13)
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_LacZ+Term_15(08/09/13)
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_AmilCP+Term_9(02/09/13)
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_AmilCP+Term_12(02/09/13)
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Degestion product quantification
- We used the nano drop to measure the DNA in 260nm and we found its concentration
| name | fnr(repressor)dig EcoilI and speI | fnr nark dig EcoilI and speI |
| conc. | 25.0ng/µl | 13.6ng/µl |
2 -Gel electrophoresis of Degestion product of promoter fnr(activator).
| [[]] |
|
3 -Gel electrophoresis of Degestion product of RBS_AmilCP+Term and RBS_LacZ+Term.
| [[]] |
|
1 - Clony PCR of Gibson assembly.
- AA 1-8 Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3
- AB 1-8 Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate
- AC 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
- AD 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
- AE 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
- AF 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate
- 4 Colonies from purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
Pick up single clone dip in 10µL of H2O in PCR tube .
Prepare PCR MIX for 55 tubes:
- Buffer Dream Taq : 137.5µL
- dNTP : 27.5µL
- Oligo 44 : 27.5µL
- Oligo 43: 27.5µL
- Dream Taq : 11µL
- H2O : 1.144mL
Add 2µL bacteria into 23µL PCR MIX in PCR tube PCR Program
| Previous week | Back to calendar | Next day |
Notebook : August 28
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize Bba_K1155000 and Bba_K1155004, Bba_K1155005, Bba_K1155006
1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions
XiaoJing
|
Purification of 08/28/13 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. We will streak these colonies again. |
We streak :
- Pfnr with RBS-Amil CP-Term in PSB1C3 with O2 at 37°C
- Pfnr with RBS-Amil CP-Term in PSB1C3 without O2 at 37°C
- Pfnr with RBS-Amil CP-Term in PSB1C3 with O2 at 30°C
- Pfnr with RBS-Amil CP-Term in PSB1C3 without O2 at 30°C
- NirB with RBS-LacZ-Term in PSB1C3 with O2 with Xgal at 37°C
- NirB with RBS-LacZ-Term in PSB1C3 without O2 with Xgal at 37°C
We also purify Pfnr with RBS-Amil CP-Term in PSB1C3 in liquid culture at 37°C using :
- Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic conditions :
- LB : 10 mL
- Clone : ...
- Pfnr with RBS-Amil CP-Term in PSB1C3 in anaerobic conditions :
- LB : 50mL
- Clone : ...
2 - Digestion of Bba_K1155000, Bba_K1155003, Bba_K1155007 by Xbal/PstI
XiaoJing
We used clone 9 and 12 for Bba_K1155003, clone 10, 11 and 15 for Bba_K1155007
Used quantities :
- Bba_K1155003, Bba_K1155007
- DNA : 14µL
- Buffer FD : 2µL
- XbaI FD : 2µL
- PstI FD : 2µL
- Bba_K1155000 :
- DNA : 5µL
- Buffer FD : 2µL
- SpeI FD : 2µL
- PstI FD : 2µL
- H2O : 9µL
We let the digestion 30 minutes at 37°C.
3 - Electrophoresis to check the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI
XiaoJing
| [[]] |
|
Expected sizes :
- RBS-LacZ-Term :
- RBS-Amil CP-Term :
| [[]] |
|
Expected sizes :
- Pfnr :
|
We obtain fragments at the right size. We will purify them. |
4 - Gel purification of the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI
XiaoJing
Protocol : Gel purification
Nanodrop :
- Pfnr : 26.2ng/µL
5 - Electrophoresis to check the gel purification of the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI
Damir
| [[]] |
|
Expected sizes :
- RBS-LacZ-Term :
- RBS-Amil CP-Term :
|
We obtain fragments at the right size for RBS-LacZ-Term. We will ligate it. |
5 - Electrophoresis to check the digestion of Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI
XiaoJing
| [[]] |
|
Expected sizes :
- NarK, NarG, NirB : ...
|
We obtain fragments at the right size for NarK. We will ligate it |
((((((((((((((====Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3====
1 - Digestion of Bba_J04450 by EcoRI/PstI
Anaïs
Used quantities :
- Buffer FD: 2µL
- H2O : 5µL
- DNA : 9µL
- EcoRI FD : 1µL
- PstI FD : 1µL
We let the digestion at 37°C during 10 minutes ??????
2 - Electrophoresis to check the digestion of Bba_J04450 by EcoRI/PstI
XiaoJing
| [[]] |
|
expected sizes :
- PSB3K3 :
|
We obtain fragments at the right size. We will ligate it. |
3 - Gel purification of the digestion of Bba_J04450 by PstI/SpeI
XiaoJing
Protocol : Gel purification
Nanodrop :
- Pfnr : 7.2ng/µL)))))))))))))))))))))))
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FRN and BphR2 proteins
1 - Colony PCR of FNR, RBS-FNR and RBS-BphR2 in DH5α
XiaoJing
|
Transformation of 08/28/13 works. We will do a Colony PCR. |
COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.
Used quantities :
- DNA : 2µL
- Mix : (it was divided in 8 tubes for 8 different colonies for each assembly with 23µL of mix in each tube. We do it twice.)
- Oligo 43 : 27.5µL
- Oligo 44 : 27.5µL
- dNTP : 27.5µL
- Buffer Dream Taq : 137.5µL
- Dream Taq : 11µL
- H2O : 1144µL
