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Team:Paris Saclay/Notebook/August/26
From 2013.igem.org
Notebook : August 26
summary
- started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoRI and PstI.
- Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which was created by iGEM UCL team in 2009
- Designed oligonucleotides for the gene coding for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group
lab work
- A.aero/anaerobic regulation system
- 1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3
- Digestion for promoter fnr(repressor and activator) in PSB1C3
- 2 enzymes SPE I and PST I
- For promoter fnr(repressor) :
| DNA | 14µl |
| SPE I | 2µl |
| PST I | 2µl |
| H2O | 0µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)nirB clone 6:
| DNA | 14µl |
| SPE I | 2µl |
| PST I | 2µl |
| H2O | 0µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)nark clone 6 :
| DNA | 4µl |
| SPE I | 2µl |
| PST I | 2µl |
| H2O | 10µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)nirB clone 7 :
| DNA | 14µl |
| SPE I | 2µl |
| PST I | 2µl |
| H2O | 0µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)narG clone 6 :
| DNA | 7µl |
| SPE I | 2µl |
| PST I | 2µl |
| H2O | 7µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)narG clone 7 :
| DNA | 14µl |
| SPE I | 2µl |
| PST I | 2µl |
| H2O | 0µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)nark Digested SPE I :
| DNA | 9µl |
| SPE I | 0µl |
| PST I | 2µl |
| H2O | 7µl |
| buffer | 2µl |
| total | 20µl |
- For promoter fnr(activator)narB Digested SPE I :
| DNA | 13µl |
| SPE I | 0µl |
| PST I | 2µl |
| H2O | 3µl |
| buffer | 2µl |
| total | 20µl |
For promoter fnr(activator)narG Digested SPE I :
| DNA | 10µl |
| SPE I | 0µl |
| PST I | 2µl |
| H2O | 6µl |
| buffer | 2µl |
| total | 20µl |
- Ligation
- After 3h of digestion, we mixed the digestion products:
| PCR product | 30µl |
| PSB1C3 | 4µl |
| Ligation buffer | 2µl |
| H2O | 14µl |
- Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
| mixture | control |
| 2µl ligation buffer | 2µl ligation buffer |
| 1µl ligase T4 | 1µl ligase T4+2µl PSB1C3 |
| 17µl H2O | 15µl H2O |
- The incubation was during 1H30.
- B.PCBs sensor system
- 1.BioBrick BphR2(regulator) in plasmid PSB1C3
- 1.BioBrick BphR2(regulator) in plasmid PSB1C3
- Using software gene manager to find the oligopeptide for amplification of BphR2.
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