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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Sep
From 2014.igem.org
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September |
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- FumA
- This week we assembled FumA with different constitutive promotors
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2070 bp and ~1800 bp) for pSB1C3_T7_FumA
- BioBrick Assembly (Suffix)
- Transformation of all constructs with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp) for all constructs
- Plasmid isolation of the positive colonies
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2070 bp and ~1800 bp)
- ccm
- Gibson Assembly with ccm and pSB1C3
- Transformation of pSB1C3_ccm with electrocompotetent cells
- GSU 3274
- This week we assembled GSU 3274 with different promotors
- BioBrick Assembly (Suffix)
- Transformation of all constructs with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55°C
- Bands as expected (~ bp) for pSB1C3_ptac and pSB1A2_T7
- Electrobiochemical reactor system
- This week we arranged the complete H-cell reactor for the first time to start first trials. Therefore we prepare the H-cell buffers and the Nafion® membrane which ensures the division of the two compartments.
- NAD/NADH Assay
- This week we startet the first test with the Promega NAD/NADH-Glo™ Assay to evaluate later cultivations respectively.
- frd (E.coli)
- This week we transform pSB1C3_T7_frd with different E.coli strains for the characterization of it respectively
- Transformation of pSB1C3_T7_frd with electrocompotetent cells of KRX ΔdcuB::oprF, JW4084-1 as well as JW0506-1 from the KEIO collection
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp)
- Restriction digestion of all constructs with EcoRI and PstI
- Bands (not) as expected (~... bp)
- Expression of frd (E.coli) for SDS-Page
- Induction was carried out with 0.1 % Rhamnose and 1 mM IPTG
- For the release of the periplasmatic protein fraction a cold osmotic shock was performed
- ccm
- Plasmid isolation of pSB1C3_ccm
- Restriction digestion with NotI
- Bands not as expected (~6611 bp)
- frd (E. coli)
- This week we removed the first illegal restriction site (PstI at 144 bp)
- PCR amplification (frd_cut1
- Annealing temperature: 55 °C
- Bands as expected (~... bp)
- Gibson Assembly of PCR products and DpnI digestion to remove the template
- Transformation with electrocompotetent cells
- Plasmid isolation for a restriction digestion with PstI afterwards
- PCR amplification (frd_cut1
- ccm
- This week we tried to get the ccm into the correct backbone
- Gibson Assembly with ccm and pSB1C3
- Transformation with electrocompotetent cells and Transformation with chemocompetent cells
- Plasmid isolation of ccm and Restriction digestion with EcoRI and PstI
- Bands mainly not as expected (~6281 bp and ~2070 bp ) because additional bands are visible.
- Therefore a restriction digestion with EcoRV and HindIII was done
- Bands not as expected because only two bands were visible
- Colony PCR with new primer(ccm_con1, VR-Primer)
- Annealing temperature: 55°C
- Bands as expected (~880 bp)
- Plasmid isolation of pSB1C3_ccm and restriction digestion with EcoRI and PstI
- Bands not as expected (~6281 bp and ~2070 bp)
- PCR amplification of ccm backbone(pSB1C3_pre_ccm, pSB1C3_suf_ccm)
- Annealing temperature: 61°C
- Bands as expected (~6281 bp)
- Electrobiochemical reactor system
- This week we performed several cyclic voltammetric measurements to characterize different electrode materials and mediators. Therefore the cathode space and anode space were filled with the appropriate buffers and the system was purged with nitrogen to remove any oxygen.
- measurements were performed under the following conditions and the the following experimental setup
- Working electrode: platinic plate or graphite electrode
- Counter electrode: platinic wire
- Reference electrode: Ag/AgCl- electrode
- Scan rate: 10 mV/s
- Scan Limit: was varied among the different measurements
- Cycle number: 3, 10 or 500
- Mediators: neutral red or bromphenol blue
- Temperature: 37°C
- FumA
- This week we removed the the mutation of FumA by PCR
- PCR amplification of FumA using a gradient(FumA_mut_fwd, FumA_mut_rev)
- Annealing temperature: 50°C to 60°C
- Bands as expected (~4000 bp)
- Gibson Assembly with FumA and pSB1C3 and digestion with DpnI to remove the template
- Transformation with chemocompetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55°C
- Bands as expected (~2100 bp)
- Plasmid isolation of pSB1C3_FumA
- Sequencing of pSB1C3_FumA (Primer: FumA_seq) confirmed the correct construct
- FumBCD
- This week we amplified and transformed FumBCD
- PCR amplification of FumBCD of pJet-Vector (FumBCD_fwd, FumBCD_rev)
- Annealing temperature: 55°C
- Bands as expected (1579 bp)
- PCR products were purified out of the gel
- Gibson Assembly with FumBCD and pSB1C3
- Digestion with DpnI to remove the template
- Transformation with electrocompotetent cells and Transformation with chemocompetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55°C
- Bands not as expected (~1909 bp)
- Deletion of dcuB and integration of oprF into chromosome
- Controll of the strain KRX ΔdcuB::oprF with and without frd (E.coli)
- FumBCD
- This week we amplified and transformed FumBCD
- PCR amplification of FumBCD of pJet-Vector (FumBCD_fwd, FumBCD_rev)
- Annealing temperature: 55°C
- Bands as expected (1579 bp)
- PCR products were purified out of the gel
- PCR amplification of FumBCD backbone(pSB1C3_pre_Fum_BCD, pSB1C3_suf_Fum_BCD)
- Annealing temperature: 55°C
- Bands (not) as expected (~2070 bp)
- Gibson Assembly with FumBCD and pSB1C3
- Transformation with electrocompotetent cells and Transformation with chemocompetent cells
- Colony PCR (FumBCD_fwd, FumBCD_rev)
- Annealing temperature: 55°C
- Bands as expected (~1579 bp)
- Plasmid isolation of pSB1C3_FumBCD
- Restriction digestion with EcoRI and PstI
- Bands not as expected (~1579 bp and ~2070 bp)
- Cloning by restriction and ligation
- PCR amplification of FumBCD of pJet-Vector (FumBCD_fwd, FumBCD_rev)
- Annealing temperature: 55°C
- Bands as expected (1579 bp)
- Restriction digestion of FumBCD with XbaI and SpeI
- Restriction digestion of the backbone with XbaI and SpeI
- Ligation of both fragments and Transformation with electrocompotetent cells as well as Transformation with chemocompetent cells
- Colony PCR (FumBCD_fwd, FumBCD_rev)
- Annealing temperature: 55°C
- Bands as expected (~1579 bp)
- Verification of the FumBCD insertion in pSB1C3
- PCR amplification (VF-Primer, FumBCD_rev)
- Annealing temperature: 55°C
- Bands as expected (~1694 bp)
- Restriction digestion of FumBCD with EcoRI and PstI
- Bands as expected (~1579 bp)
- anaerobic cultivation for characterization of pSB1C3_T7_frd
- We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_frd
- Media
- M9 with xylose (50 mM)
- M9 with xylose (50 mM) and fumarate (50 mM)
- M9 with fumarate (50 mM)
- induction was performed by adding 0,1% Rhamnose
- To document the growth curve and the consumption of substrate daily samples were taken for OD600 measurement and HPLC analytics
- anaerobic cultivation for characterization of the antiporter δdcuB
- We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_frd
- Strains
- Media
- M9 with xylose (50 mM)
- M9 with xylose (50 mM) and fumarate (50 mM)
- M9 with fumarate (50 mM)
- induction was performed by adding 0,1% Rhamnose
- To document the growth curve and the consumption of substrate daily samples were taken for OD600 measurement and HPLC analytics
