Team:Paris Saclay/Notebook/August/28
From 2013.igem.org
(→2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α) |
(/* 1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in pSB1C3, NirB with RBS-Amil CP-Term in pSB1C3, Pfnr with RBS-LacZ-Term in pSB1C3, Pfnr with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions) |
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* NirB with RBS-Amil CP-Term in pSB1C3 with O2 | * NirB with RBS-Amil CP-Term in pSB1C3 with O2 | ||
* NirB with RBS-Amil CP-Term in pSB1C3 without O2 | * NirB with RBS-Amil CP-Term in pSB1C3 without O2 | ||
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+ | ===='''2 - Culture of mutant strain MG1655Z1 Δfnr''' ==== | ||
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+ | XiaoJing | ||
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+ | {| | ||
+ | | style="border:1px solid black;padding:5px;background-color:#DE;" | | ||
+ | Transformation of 08/27/13 works. We will do a purification on LB plates. | ||
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+ | We streaked our colonies on plates with LB and incubated them at 42°C. | ||
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''=== | ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''=== |
Revision as of 20:20, 4 October 2013
Notebook : August 28
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize BBa_K1155000 and BBa_K1155004
1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in pSB1C3, NirB with RBS-Amil CP-Term in pSB1C3, Pfnr with RBS-LacZ-Term in pSB1C3, Pfnr with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions
XiaoJing
Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in pSB1C3, Pfnr with RBS-LacZ-Term in pSB1C3, Pfnr with RBS-Amil CP-Term in pSB1C3. |
We streak colonies from construction :
- Pfnr with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
- Pfnr with RBS-Amil CP-Term in pSB1C3 with O2
- Pfnr with RBS-Amil CP-Term in pSB1C3 without O2
- NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal
- NirB with RBS-Amil CP-Term in pSB1C3 with O2
- NirB with RBS-Amil CP-Term in pSB1C3 without O2
2 - Culture of mutant strain MG1655Z1 Δfnr
XiaoJing
Transformation of 08/27/13 works. We will do a purification on LB plates. |
We streaked our colonies on plates with LB and incubated them at 42°C.
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FRN and BphR2 proteins
1 - Gel purification of pSB1C3 digested by DnpI
XiaoJing
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :
- pSB1C3 : 37.2ng/µL
2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI
XiaoJing
]] |
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expected sizes :
- pSB1C3 : 2070 bp
We obtain fragment at the right size. The gel purification was good. We will use it for Gibson assembly. |
3 - Gibson assembly
XiaoJing
Used quantities :
- RBS-BphR2 :
- pSB1C3 : 3µL
- BphR2 Part I : 1µL
- BphR2 Part II : 1µL
- Gibson mix : 15µL
- FNR :
- pSB1C3 : 3µL
- FNR Part I : 1µL
- FNR Part II : 1µL
- Gisbon mix : 15µL
- RBS-FNR :
- pSB1C3 : 3µL
- RBS-FNR Part I : 1µL
- FNR Part II : 1µL
- Gibson mix : 15µL
We incubate these mix at 50°C during 1h inside PCR machine.
2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain
XiaoJing
Protocol : Bacterial transformation
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