Team:Paris Saclay/Notebook/August/28
From 2013.igem.org
Notebook : August 28
summary
- We got purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
so we do a purify of 4 clonies in two plate.incubated at 37°C in aerobic and anaerobic condition over night to verify the different.
- Prepare LB plates with Chlorenphenicol and Xgal .Pufify single conle of promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 and promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 to see if color will change.
- Gel extration of Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and do Gibson assembly again .
lab work
A - Aerobic/Anaerobic regulation system / B - PCB sensing system
- 1 - Gel extraction of the PSB1C3 Clean for Gibson.
Protocol : Gel extraction
- 2- Migration of 3µl PSB1C3 Clean for Gibson (product gel extraction) '
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- 3- product quantification.
- We used the nano drop to measure the DNA in 260nm and we found its concentration
name | PSB1C3 Clean dig Dnp1 gel extraction |
conc. | 37.2ng/µl |
- 4 - Gibson assembly.
- RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
- FNR_part1, FNR part1 and plasmid PSB1C3
- RBS_FNR part1, FNR_part2 and plasmid PSB1C3
Sample Volume:
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These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
A - Aerobic/Anaerobic regulation system
- 5- Purify single clone of B5 promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night
- 7- Purify single clone of C1, C2, C5 ,C6promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night
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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 :
- Pfnr with RBS-LacZ-Term in PSB1C3 with O2 and Xgal
- 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
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 : 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 frragment 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 let these mix at 50°C during 1h.
2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α
XiaoJing
Protocol : Bacterial transformation