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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul
From 2014.igem.org
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July |
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- FumA
- This week we amplified the pSB1C3 backbone of FumA to use it for Gibson Assembly afterwards
- PCR amplification of FumA ( pSB1C3_pre_Fum_A, pSB1C3_suf_Fum_A )
- Annealing temperature: 55 °C
- Bands are as expected (2070 bp)
- FumBCD
- This week we amplified the pSB1C3 backbone of FumBCD to use it for Gibson Assembly afterwards
- PCR amplification ( pSB1C3_pre_Fum_BCD, pSB1C3_suf_Fum_BCD )
- Annealing temperature: 55 °C
- Bands are as expected (2070 bp)
- PCR purification of backbones
- deletion of dcuB and integration of oprF into chromosome
- This week we started the deletion of the fumarate/succinate antiporter dcuB and the integration of outer membrane porin oprF into chromosome using the Genebridge Red/ET-System
- pRedET plasmid
- Plasmid isolation of pRedET plasmid
- oprF (BBa_K1172507)
- Plasmid isolation of pSB1C3_BBa_K1172507
- deletion of dcuB and integration of oprF into chromosome
- This week we amplified the pR6K-cassette and the oprF gene to connect them to each other. Additionally we transform the RedET plasmid into the E. coli strain KRX.
- RedET plasmid
- Transformation with electrocompotetent cells of the RedET plasmid into E. coli KRX
- pR6K
- PCR amplification of pR6K-cassette (pR6K_dcuB_fwd, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands are as expected (~1600 bp) but slightly visible
- PCR amplification of pR6K-cassette is repeated two times
- PCR product pR6K was purified out of the gel
- oprF (BBa_K1172507)
- PCR amplification of oprF for integration into chromosome ( porine_fwd_FRT, porine_rev_FRT )
- Annealing temperature: 55 °C
- bands are as expected (~1359 bp)
- PCR product oprF was purified out of the gel
- Connection of pR6K and oprF
- PCR amplification to connect the pR6K-cassette and oprF (porine_fwd_FRT, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands not as expected because of too much template (~2900 bp)
- Next time the PCR amplification was planned with a different usage of primer
- additionally a new PCR amplification (as described further up) of pR6K-cassette and a new purification of it and oprF out of the gel was done
- PCR amplification to connect the pR6K-cassette and oprF was repeated with the new purified pR6K-cassette and oprF and different primer usage
- PCR amplification was initially performed 15 cycles without primer and afterwards 30 cycles with addition of primer
- deletion of dcuB and integration of oprF into chromosome
- connection of pR6K-cassette and oprF
- PCR amplification to connect the pR6K-cassette and oprF
- PCR amplification and purification of pR6K-cassette and oprF seperatly
