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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)
- 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
