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Team:Tec-Monterrey/projectresults/methods
From 2011.igem.org
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| - | + | SacC gene from <i> Zymomonas mobilis </i> was PCR amplified with the primers S1PSF: 5’-GAATT CGCGG CCGCT TCTAG AGGAG CTCAT GTTTA ATTTT AATGC CAGTC GC-3’, S1PSR 5’-CTGCA GCGGC CGCTA CTAGT AGCTA GCGTA TTTGC GACGA TCAGG G-3’. The amplified fragment was cloned in pGEM T Easy Vector. The amplification mixture for 50 mL contained 1U of Platinum Taq HF polymerase (Invitrogen), 60 mM Tris-SO<sub>4</sub>, 18 mM Ammonium Sulfate, 0.2 mM for each dNTP, 2 mM MgSO<sub>4</sub>, 2 mM of forward and reverse primers. PCR was performed in an MultiGene (Labnet) using the following program: 94 ºC for 5 min, 35 cycles of 94 ºC for 45 s, 56.4 ºC for 30 s, and 68 ºC for 1 min, and finally an extension step at 68 ºC for 5 min. The PCR product cloned in pGEM T Easy Vector was added to the registry (BBa_K633003) and the product was cloned in the plasmid pSB1C3 and added to the registry (BBa_...TBD). | |
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| - | The ompA+sacC construction was generated by joining the biobricks of a promoter (araC BBa_I13458 and P<sub>BAD</sub> BBa_K206000), RBS (BBa_B0034), lpp+ompA (BBa_K103006) with the biobrick standard assembly protocol (Manual). The expected DNA fragment of the ompA + sacC construct was confirmed by several restriction endonuclease reactions and agarose gel electrophoresis, and used to transform the <i>E.coli</i> strins BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783. | + | The ompA+sacC construction was generated by joining the biobricks of a promoter (araC <a href="http://partsregistry.org/Part:BBa_I13458">BBa_I13458 </a>and P<sub>BAD</sub> <a href="http://partsregistry.org/Part:BBa_K206000"> BBa_K206000</a>), RBS (<a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>), lpp+ompA (<a href="http://partsregistry.org/Part:BBa_K103006">BBa_K103006</a>) with the biobrick standard assembly protocol (Manual). The expected DNA fragment of the ompA + sacC construct was confirmed by several restriction endonuclease reactions and agarose gel electrophoresis, and used to transform the <i>E.coli</i> strins BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783. |
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Revision as of 01:21, 18 October 2011
• Try another E. coli strain like XL1 Blue, C43, Bl21 SI and others
SacC gene from Zymomonas mobilis was PCR amplified with the primers S1PSF: 5’-GAATT CGCGG CCGCT TCTAG AGGAG CTCAT GTTTA ATTTT AATGC CAGTC GC-3’, S1PSR 5’-CTGCA GCGGC CGCTA CTAGT AGCTA GCGTA TTTGC GACGA TCAGG G-3’. The amplified fragment was cloned in pGEM T Easy Vector. The amplification mixture for 50 mL contained 1U of Platinum Taq HF polymerase (Invitrogen), 60 mM Tris-SO4, 18 mM Ammonium Sulfate, 0.2 mM for each dNTP, 2 mM MgSO4, 2 mM of forward and reverse primers. PCR was performed in an MultiGene (Labnet) using the following program: 94 ºC for 5 min, 35 cycles of 94 ºC for 45 s, 56.4 ºC for 30 s, and 68 ºC for 1 min, and finally an extension step at 68 ºC for 5 min. The PCR product cloned in pGEM T Easy Vector was added to the registry (BBa_K633003) and the product was cloned in the plasmid pSB1C3 and added to the registry (BBa_...TBD).
The ompA+sacC construction was generated by joining the biobricks of a promoter (araC BBa_I13458 and PBAD BBa_K206000), RBS (BBa_B0034), lpp+ompA (BBa_K103006) with the biobrick standard assembly protocol (Manual). The expected DNA fragment of the ompA + sacC construct was confirmed by several restriction endonuclease reactions and agarose gel electrophoresis, and used to transform the E.coli strins BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783.
The E. coli strains containing the ompA+sacC construct and non-trasnsformed strains as negative controls were cultured with 6 mL of media M9 with glycerol as carbon source at an initial optical density at 600 nm (OD600) of 0.1. The batch cultures were performed at 37C until the OD600 of 0.6 was attained. Then, the expression was induced with 0.1mM of L-arabinose and the temperature of postinduction was changed to 15C. Culture samples collected from the bioreactor were harvested by centrifugation. The half volume was used for the whole cell assay and the other half was processed with Clontech x-Tractor kit to obtain soluble and insoluble fraction of each strain. Both fractions were separated by 10% SDS-PAGE and visualized with GelCode Blue Stain Reagent (Thermo).
