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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun
From 2014.igem.org
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June |
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- Transformation of electrocompotetent E.coli KRX cells:
- K538742: pSB1C3-kivD (parts distribution from 2013, plate 1, 24L)
- K539621: pSB1C3-ilvC (parts distribution from 2013, plate 1, 24F)
- K539626: pSB1C3-ilvD (parts distribution from 2013, plate 1, 22J)
- K539627: pSB1C3-alsS (parts distribution from 2013, plate 1, 24H)
- Resulting colonies were selected to start liquid cultures:
- 5 ml of LB in 20 ml reaction tube
- incubation over night at 37°C and 250 rpm
- Plasmid isolation by ThermoScientific MiniPrep
- Sanger sequencing using VF and VR as sequencing primers
- Identities of all parts were confirmed, but not whole sequence of the parts was covered
- Glycerin stocks were created for all four strains:
- E. coli KRX pSB1C3-alsS
- E. coli KRX pSB1C3-ilvC
- E. coli KRX pSB1C3-ilvD
- E. coli KRX pSB1C3-kivD
- pSB1C3-alsS-ilvC-ilvD-kivD
- PCR amplification of all four coding sequences for a Gibson Assembly
- Elongation time: 90 seconds
- Annealing temperature: 55°C
- Combinations of primer and template combinations are listed in the table below. Sequences for Gibson Assembly are introduced by the primers. Additionally the forward primers contain a RBS (BBa_B0034).
- Products were analyzed by agarose gel electrophoresis
- PCR conditions need to be optimized do to low product quality and missing products
| forward primer | reverse primer | template |
|---|---|---|
| fw_pSB1C3_alsS | rv_ilvC_alsS | pSB1C3-alsS |
| fw_alsS_ilvC | rv_ilvD_ilvC | pSB1C3-ilvC |
| fw_ilvC_ilvD | rv_kivD_ilvD | pSB1C3-ilvD |
| fw_ilvD_kivD | rv_pSB1C3_kivD | pSB1C3-kivD |
| fw_kivD_pSB1C3 | rv_alsS_pSB1C3 | pSB1C3-CFP |
