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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug
From 2014.igem.org
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August |
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- Deletion of dcuB and integration of oprF into chromosome
- This week we amplified and purified the pR6K-cassette again in order to connect it to oprF
- pR6K
- PCR amplification of pR6K-cassette
- Plasmid isolation of pR6K-cassette and Purification out of the gel
- PCR amplification to connect the pR6K-cassette and oprF
- Bands as expected (~3300 bp)
- PCR product was purified out of the gel
- Fum A
- This week we assembled FumA it with the T7-Promotor as well as with several Anderson-Promotor using Biobrick-Assembly
- BioBrick Assembly (Suffix)
- BioBrick Assembly (Suffix)
- Cultivation of E.coli with neutral red and bromphenol blue
- This week we tested if neutral red and bromphenol blue have an influence on the growth of E. coli. Therefore we cultivated E.coli wildtype in M9 medium with glucose supplemented with 100 µM of neutral red or bromphenol blue. Samples were taken for OD600 measurement to document the growth curve.
- Deletion of dcuB and integration of oprF into chromosome
- This week we checked our first colonies to verify the deletion of dcuB
- pR6K
- The pR6K-cassette for the dcuB deletion was purified out of the gel
- Transformation of pR6K-cassette for the deletion of dcuB with electrocompotetent cells
- pRedET
- Transformation of RedET plasmid with electrocompotetent cells
- dcuB
- Colony PCR to verify the deletion of dcuB (dcuB_del_kon1, dcuB_del_kon2)
- Annealing temperature: 55 °C
- Bands not as expected (~2390 bp)
- Fum A
- This week we assembled FumA with different promotor
- Transformation of
- BBa_J23101_FumA
- BBa_J23102_ Fum A
- BBa_J23104_FumA
- pSB1A2_T7 with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Band as expected (~2200 bp)
- Plasmid isolation of BBa_J23102_ Fum A
- Sequencing of
- BBa_J23101_FumA
- BBa_J23102_FumA
- BBa_J23104_FumA
- pSB1A2_T7_FumA showed different mutations
- Fum BCD
- This week wie amplified FumBCD and its backbone
- PCR amplification of Fum BCD-backbone (pSB1C3_pre_Fum_BCD , pSB1C3_pre_Fum_BCD)
- Annealing temperature: 55 °C
- Bands as expected (2070 bp)
- The Fum BCD backbone was purified out of the gel
- PCR amplification of Fum BCD (Fum_BCD_fwd , Fum_BCD_rev)
- Annealing temperature: 55 °C
- Bands not as expected (1579 bp)
- ccm
- This week we amplified the ccm-cassette and its backbone
- PCR amplification of the ccm-backbone (pSB1C3_suf_ccm, pSB1C3_pre_ccm)
- Annealing temperature: 65 °C
- Bands as expected (~2070 bp) but slightly visible because of inappropriate annealing temperature
- PCR amplification was repeated with an annealing temperature of 55 °C
- Both PCR products were purified out of the gel
- PCR amplification of ccm-cassette using a gradient (ccm_fwd, ccm_rev)
- Annealing temperature: 61 °C appeared as the optimum
- Bands as expected (~6281 bp)
- PCR amplification of ccm-cassette (ccm_fwd, ccm_rev)
- Annealing temperature: 61 °C
- Bands as expected (~6281 bp)
- PCR product was purified
- GSU 3274
- This week we amplified the GSU 3274 cytochrome and its backbone
- PCR amplification of GSU 3274-backbone (pSB1C3_pre_pccH, pSB1C3_suf_pccH)
- Annealing temperature: 65 °C
- Bands not as expected (~2070 bp) because of inappropriate annealing temperature
- PCR amplification was repeated with an annealing temperature of 55 °C
- Bands as expected (~2070 bp)
- PCR amplification of GSU 3274 using a gradient (pccH_fwd, pccH_rev)
- Annealing temperature: 55 °C appeared as the optimum
- Bands as expected (~453 bp)
- PCR amplification of GSU 3274 (pccH_fwd, pccH_rev)
- Annealing temperature: 55 °C
- Bands as expected (~453 bp)
- genomic DNA of G. sulfurreducens as well as the PCR product of the gradient-PCR was used as template
- PCR product was purified
- Deletion of dcuB and integration of oprF into chromosome
- This week we contninued to test colonies for the verification of the deletion if dcuB
- Connection of pR6K-cassette and oprF
- PCR amplification to connect the pR6K-cassette and oprF
- Bands as expected (~3300 bp)
- PCR product was purified out of the gel
- Colony PCR to verify the deletion of dcuB (dcuB_del_kon1, dcuB_del_kon2)
- Annealing temperature: 55 °C
- Bands not as expected (~4046 bp)
- PCR amplification to connect the pR6K-cassette and oprF
- Connection of pR6K-cassette and oprF
- Fum A
- This week wie tested some colonies for FumA
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (1843 bp)
- frd (E. coli)
- This week we amplified and transformed frd (E. coli)
- PCR amplification (T7_frd_Ec_fwd, T7_frd_Ec_rev)
- Annealing temperature: 55 °C
- Bands as expected (~3300 bp)
- Additionally there were undefined bands at ~1200 bp
- illiegal restriction sites were not removed yet
- Gibson Assembly with frd (E. coli) and pSB1C3
- Transformation with electrocompotetent cells of pSB1C3_frd
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~3600 bp)
- ccm
- This week we transformed the ccm-cassette and tested colonies for it
- DpnI-digest of ccm-backbone
- Gibson Assembly with ccm and pSB1C3
- Transformation with electrocompotetent cells of pSB1C3_ccm
- Colony PCR of pSB1C3_ccm (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~6611 bp) probably because it was not completely amplified because of its length
- To check whether one of the colonies is positiv a restriction digestion was done
- Restriction digestion with EcoRI and PstI
- Bands not as expected (~2070 bp and 6281 bp)
- GSU 3274
- This week we transformed the GSU 3274 and tested colonies for it
- DpnI-digest of GSU 3274-backbone
- Gibson Assembly with GSU 3274 and pSB1C3
- Transformation with electrocompotetent cells of pSB1C3_GSU 3274
- Colony PCR of pSB1C3_GSU 3274 (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~783 bp)
- Restriction digestion with EcoRI and PstI
- Bands not as expected (~2070 bp and 453 bp)
- Deletion of dcuB and integration of oprF into chromosome
- This week we tested the next colonies for the deletion of dcuB and performed the NPN-assay to verify the integration of oprF into chromosome
- oprF
- NPN-uptake assay for porine verification
- dcuB
- Colony PCR to verify the deletion of dcuB (dcuB_del_kon1, dcuB_del_kon2)
- Annealing temperature: 55°C
- Bands as expected (~4046 bp)
- another Colony PCR to verify the positive colony
- FumA
- This week we assembled FumA with different promotor
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- BBa_J23101
- BBa_J23102
- BBa_J23104
- pSB1A2_T7
- Insert (digested with XbaI, PstI)
- FumA
- Transformation of all contrsucts with electrocompotetent cells
- Colony PCR with all contructs (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp) for BBa_J23101_FumA and pSB1A2_T7_FumA
- frd (E. coli)
- This week we transformed frd and checked several colonies for it
- DpnI digest of Gibson-Assembly of frd (E. coli) to remove the template
- Transformation of Gibson-Assembly with electrocompotetent cells
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp) but there were several other bands too
- Therefore another Transformation with electrocompotetent cells was done
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~ 3600 bp)
- PCR amplification of frd backbone (pSB1C3_frd_pre, pSB1C3_frd_suf)
- Annealing temperature: 55 °C
- Bands as expected (~ 2070 bp)
- frd backbone was purified out of the gel
- Gibson Assembly with frd and pSB1C3
- Transformation of pSB1C3_frd with chemocompetent cells
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~ 3600 bp)
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2070 bp and 3300 bp)
- Plasmid isolation of pSB1C3_frd
- sequencing of pSB1C3_frd confirmed the correct construct
- next the illegal restriction sites had to be removed
- ccm
- This week we performed Eckhardt gels as a new method besides Colony PCR to identify positiv colonies
- Eckhardt gel of ccm was performed two times
- Bands not impeccable to identify (~6230 bp) and therefore a restriction digestion was done
- Bands not as expected (~2070 bp and ~6239 bp)
- GSU 3274
- This week we tested colonies for GSU 3274
- Colony PCR of GSU 3274 (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~783 bp)
- Plasmid isolation of GSU 3274
- Restriction digestion with PstI and EcoRI
- Bands as expected (~20170 bp and 453 bp)
