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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Sep
From 2014.igem.org
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September |
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- adhA
- This week we tried to verify the positive cutures that were identified last week by a restriction digest
- Plasmid isolation of pSB1K3_adhA
- Restriction digestion with EcoRI and PstI of pSB1K3_adhA
- Bands as expected (~1200 bp)
- Successful sequencing
Agarose gel from the restriction digestion with EcoRI and PstI. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- T7_alsS_ilvC_ilvD_kivD
- This week we tried to verify the positive cutures that were identified last week by a restriction digest
- Plasmid isolation of pSB1A2_T7_alsS_ilvC_ilvD_kivD
- Restriction digestion with EcoRI and PstI of pSB1A2_T7_alsS_ilvC_ilvD_kivD
- Bands not as expected (Backbone: ~2200 and insert:~7500bp)
- pSB1K3_alsS_ilvC_ilcD_kivD_adhA
- This week we tried to combine the pSB1K3_alsS_ilvC_ilvD_kivD with adhA
- BioBrick Assembly (Suffix)
- Colony PCR (fw-ilvD-kivD, rev-pSB1C3-adhA)
- Annealing temperature: 65 °C
- Bands not as expected (~ 3800 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD
- This week we tried to reclone alsS_ilvC_ilvD_kivD into pSB1C3
- Restriction digestion of pSB1K3_alsS_ilvC_ilvD_kivD and pSB1C3_RFP with EcoRI and PstI
- Bands as expected
(~ 7500 bp for alsS_ilvC_ilvD_kivD and ~ 2200 bp for pSB1C3) - Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvD_kivD, rv_pSB1C3_kivD)
- Annealing temperature: 65 °C
- Bands not as expected (~1800 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD_adhA
- This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly
- Colony PCR (fw_ilvD_kivD , rev_pSB1C3_adhA)
- Annealing temperature: 65°C
- Bands not as expected (~3000 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD
- This week we tried to reclone alsS_ilvC_ilvD_kivD into pSB1C3
- Restriction digestion of pSB1K3_alsS_ilvC_ilvD_kivD and pSB1C3_RFP with EcoRI and PstI
- Bands as expected
(~ 7500 bp for alsS_ilvC_ilvD_kivD and ~ 2200 bp for pSB1C3) - Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvD_kivD, rv_pSB1C3_kivD)
- Annealing temperature: 65 °C
- Bands not as expected (~1800 bp)
- pSB1C3_adhA
- This week we tried to reclone adhA into pSB1C3
- Restriction digestion of pSB1K3_adhA and pSB1C3_RFP with EcoRI and PstI
- Bands as expected
(~ 1200 bp for adhA and ~ 2200 bp for pSB1C3) - Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands not as expected (~ 1200 bp)
- pSB1A2_T7_adhA
- This week we tried to contruct the pSB1A2_T7_adhA-Plasmid
- BioBrick Assembly (Suffix)
- Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands not as expected (~ 1200 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD_adhA
- This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly
- Colony PCR (fw_ilvD_kivD , rev_pSB1C3_adhA)
- Annealing temperature: 65°C
- Bands not as expected (~3000 bp)
- Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the PCR purification. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_alsS_ilvC_ilvD_kivD has an illigal restriction side.
Because of the integration of RBS's between all genes in the alsS_ilvC_ilvD_kivD-Plasmid, a restriction side between alsS and a RBS occurs.
New primers were ordered: rv_ilvC_alsS-new and fw_alsS_ilvC-new
- pSB1C3_alsS_ilvC_ilvD_kivD
- This week the new primers rv_ilvC_alsS-new and fw_alsS_ilvC-new arrived. We tried to remove the illegale restriction side.
- We tried different PCR amplifications to increase the chance to get a correct BioBrick. Therefore we amplified five parts for two combination possibilities in the Gibson Assembly:
- fw_kivD_pSB1C3, rv_alsS_pSB1C3 on pSB1C3_RFP
→ pSB1C3 with ~ 2200 bp - fw_pSB1C3_alsS, rv_ilvC_alsS-new on pSB1K3_alsS_ilvC_ilvD_kivD
→ alsS with ~ 1800 bp - rv_pSB1C3_kivD, fw_alsS_ilvC-new on pSB1K3_alsS_ilvC_ilvD_kivD
→ ilvC_ilvD_kivD with ~ 5200 bp - rv_kivD_ilvD, fw_alsS_ilvC-new on pSB1K3_alsS_ilvC_ilvD_kivD
→ ilvC_ilvD with ~ 3600 bp - fw_ilvD_kivD, rv_pSB1C3_kivD on pSB1K3_alsS_ilvC_ilvD_kivD
→ kivD with ~ 1800 bp
- Annealing temperature: 65 °C
- Bands as expected (see above)
- fw_kivD_pSB1C3, rv_alsS_pSB1C3 on pSB1C3_RFP
- PCR products were purified out of the gel
- Gibson Assembly with the previous PCR products:
- 1., 2. and 3.
- 1., 2., 4. and 5.
- Transformation with electrocompotetent cells
- Colony PCR (fw_pSB1C3_alsS, rv_ilvC_alsS-new)
- Annealing temperature: 65 °C
- Bands as expected (~1800 bp)
- Liquid cultures for a restriction digest were prepared.
- Plasmid isolation of pSB1C3-alsS-ilvC-ilvD-kivD without illigale restriction side
- Restriction digestion with XbaI and PstI
- Bands as expected (backbone: 2,2 kb and insert: 7 kb)
- Successful sequencing
- pSB1C3_adhA
- This week we tried to construct the pSB1C3_adhA using Gibson Assembly.
- PCR amplification of the pSB1C3_RFP for pSB1C3 as backbone (fw_adhA_pSB1C3, rev_adhA_pSB1C3) and pSB1K3_adhA for adhA as insert (rev_pSB1C3_adhA, fw_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands as expected (~1200 bp for the adhA and ~ 2200 bp for the backbone)
- PCR products were purified out of the gel
- Gibson Assembly with adhA and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (rev_pSB1C3_adhA, fw_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands as expected (~1200 bp)
- Liquid cultures for a restriction digest were prepared.
- Plasmid isolation of pSB1C3-adhA
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: 2,2 kb and insert: 1,2 kb)
- Successful sequencing
- pSB1C3_ptac_alsS_ilvC_ilcD_kivD
- This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the ptac promotor.
- BioBrick Assembly (Suffix and Prefix)
- Backbone (digested with SpeI, PstI)
- pSB1C3_ptac
- Insert (digested with XbaI, PstI)
- alsS_ilvC_ilvD_kivD
- Backbone (digested with EcoRI, XbaI)
- alsS_ilvC_ilvD_kivD_pSB1C3
- Insert (digested with EcoRI, SpeI)
- ptac
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, rv_ilvC_alsS-new)
- Annealing temperature: 55°C
- Bands as expected (~3000 bp)
- Liquid culture for a restriction digest was prepared.
- Plasmid isolation of pSB1C3-ptac_alsS_ilvC_ilvD_kivD
- Restriction digestion with XbaI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~9,5 kb)
- Successful sequencing
- pSB1C3_ptac_adhA
- This week we tried to combine the pSB1C3_adhA contruct with the ptac promotor.
- BioBrick Assembly (Suffix and Prefix)
- Backbone (digested with SpeI, PstI)
- pSB1C3_ptac
- Insert (digested with XbaI, PstI)
- adhA
- Backbone (digested with EcoRI, XbaI)
- adhA_pSB1C3
- Insert (digested with EcoRI, SpeI)
- ptac
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, rev_pSB1C3_adhA)
- Annealing temperature: 55°C
- Bands as expected (~2800 bp)
- Liquid cultures for a restriction digest were prepared.
- Plasmid isolation of pSB1C3-adhA
- Restriction digestion with XbaI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~2,4 kb)
- Successful sequencing
- pSB1C3_alsS_ilvC_ilcD_kivD_adhA
- This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the adhA from L. lactis.
- BioBrick Assembly (Suffix and Prefix)
- Backbone (digested with SpeI, SpeI)
- pSB1C3_adhA
- Insert (digested with EcoRI, PstI)
- alsS_ilvC_ilvD_kivD
- Backbone (digested with SpeI, PstI)
- pSB1C3_alsS_ilvC_ilvD_kivD
- Insert (digested with XbaI, PstI)
- ptac
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvD_kivD, rev_pSB1C3_adhA)
- Annealing temperature: 55°C
- Bands as expected (~3000 bp)
- Liquid culture for a restriction digest was prepared.
- Plasmid isolation of pSB1C3_alsS_ilvC_ilvD_kivD_adhA
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~9,5 kb)
- Successful sequencing
- pSB1A2_T7_adhA
- This week we tried to contruct the pSB1A2_T7_adhA-Plasmid
- BioBrick Assembly (Suffix)
- Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands as expected (~ 1200 bp)
- Liquid culture for a restriction digest was prepared.
- Plasmid isolation of pSB1A2-T7_adhA
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~1,3 kb)
- Successful sequencing
- For the protein expression analysis of AdhA we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD600 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples one, two and three and 20 hours later. Of these samples, we made a SDS Page
- GC-MS analysis of the isobutanol production
- For the analysis of the isobutanol production we decided to use a GC-MS analysis. Because we will cultivate in LB medium, we made a calibration line with LB and the following concentrations of isobutanol for further analysis and quantification of our samples.
- 0,5 % isobutanol
- 0,1 % isobutanol
- 0,05 % isobutanol
- 0,01 % isobutanol
- 0,001 % isobutanol
- Solvent extraction with 100µl LB-sample and 900µl aceton
- 5 min centrifugation
- 150 µl supernatant in the GC-MS vials for the analysis
- The detection of isobutanol works for all tested concentrations but the 2-butanol peak was to high, so we need to do this again with 0.1% 2-Butanol.
