Team:Paris Saclay/Notebook/August/28
From 2013.igem.org
(→1 - Gel purification of PSB1C3 digested by DnpI) |
(→2 - Electrophoresis to check the gel purification of pSB1C3 digested by DnpI) |
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===='''Objective : characterize BBa_K1155000 and BBa_K1155004'''==== | ===='''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, | + | ===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in pSB1C3, NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions'''==== |
XiaoJing | XiaoJing | ||
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{| | {| | ||
| style="border:1px solid black;padding:5px;background-color:#DE;" | | | style="border:1px solid black;padding:5px;background-color:#DE;" | | ||
- | 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 | + | 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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3. |
|} | |} | ||
- | We streak : | + | We streak colonies from construction : |
- | * | + | * Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal |
- | * | + | * Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 |
- | * | + | * Pndh* with RBS-Amil CP-Term in pSB1C3 without O2 |
* NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal | * 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 with O2 | ||
* NirB with RBS-Amil CP-Term in pSB1C3 without O2 | * NirB with RBS-Amil CP-Term in pSB1C3 without O2 | ||
+ | |||
+ | ===='''2 - Culture of mutant strain MG1655Z1 Δfnr''' ==== | ||
+ | |||
+ | XiaoJing | ||
+ | |||
+ | {| | ||
+ | | style="border:1px solid black;padding:5px;background-color:#DE;" | | ||
+ | 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'''=== | ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''=== | ||
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* pSB1C3 : 37.2ng/µL | * pSB1C3 : 37.2ng/µL | ||
- | ===='''2 - Electrophoresis to check the gel purification of | + | ===='''2 - Electrophoresis to check the gel purification of pSB1C3 digested by DnpI'''==== |
XiaoJing | XiaoJing | ||
{| | {| | ||
- | | style="width:350px;border:1px solid black;" |]] | + | | style="width:350px;border:1px solid black;" |[[File:Psgel12808.jpg]] |
| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
* Well 1 : 6µL DNA Ladder | * Well 1 : 6µL DNA Ladder | ||
- | * Well 2 : 3µL of | + | * Well 2 : 3µL of pSB1C3 digested by DpnI + 1µl of 6X loading dye |
* Gel : 1% | * Gel : 1% | ||
|} | |} | ||
expected sizes : | expected sizes : | ||
- | * | + | * pSB1C3 : 2070 bp |
{| | {| | ||
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" | | | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" | | ||
- | We | + | We obtained a fragment at the right size. The gel purification was good. We will use it for Gibson assembly. |
|} | |} | ||
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* RBS-BphR2 : | * RBS-BphR2 : | ||
- | ** | + | ** pSB1C3 : 3µL |
** BphR2 Part I : 1µL | ** BphR2 Part I : 1µL | ||
** BphR2 Part II : 1µL | ** BphR2 Part II : 1µL | ||
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* FNR : | * FNR : | ||
- | ** | + | ** pSB1C3 : 3µL |
** FNR Part I : 1µL | ** FNR Part I : 1µL | ||
** FNR Part II : 1µL | ** FNR Part II : 1µL | ||
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* RBS-FNR : | * RBS-FNR : | ||
- | ** | + | ** pSB1C3 : 3µL |
** RBS-FNR Part I : 1µL | ** RBS-FNR Part I : 1µL | ||
** FNR Part II : 1µL | ** FNR Part II : 1µL | ||
** Gibson mix : 15µL | ** Gibson mix : 15µL | ||
- | We | + | We incubate these mix at 50°C during 1h inside PCR machine. |
- | ===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α'''==== | + | ===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain'''==== |
XiaoJing | XiaoJing |
Latest revision as of 01:20, 5 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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* 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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3. |
We streak colonies from construction :
- Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
- Pndh* with RBS-Amil CP-Term in pSB1C3 with O2
- Pndh* 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
|
expected sizes :
- pSB1C3 : 2070 bp
We obtained a 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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