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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 | + | The 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) thermocycler 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 was first sub-cloned in pGEM T Easy Vector (Promega) and added to the registry (<a href="http://partsregistry.org/Part:BBa_K633003">BBa_K633003</a>) and then was cloned to 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 | + | The ompA+sacC construction was generated by joining the biobricks of the araC-P<sub>BAD</sub> promoter (<a href="http://partsregistry.org/Part:BBa_I13458">BBa_I13458 </a>and <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>), and sacC (<a href="http://partsregistry.org/Part:BBa_K633003">BBa_K633003</a>) with the biobrick standard assembly protocol (<a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf"> Manual</a>). The expected DNA fragment of the ompA + sacC construct was confirmed by several restriction endonuclease reactions, and used to transform the <i>E.coli</i> strins BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783. The <i>E. coli</i> strains (BL21SI, Rosetta Gami, XL1 Blue, and C43) were obtained from ¿...? and the strain BW27783 was donated by <a href="https://2010.igem.org/Team:Tec-Monterrey">Tec-Monterrey 2010</a>) |
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| - | The <i>E. coli</i> strains containing the ompA+sacC construct and non- | + | The <i>E. coli</i> strains containing the ompA+sacC construct and non-transformed strains as negative controls were cultured in 6 mL of media M9 with glycerol as its unique carbon source. The initial optical density at 600 nm (OD<sub>600</sub>) was 0.1, from there the batch cultures were incubated at 37°C until the OD<sub>600</sub> of 0.6 was attained. The expression was induced with 0.1mM of L-arabinose and the temperature of postinduction was changed to 15 °C. Culture samples collected from the bioreactor were harvested by centrifugation. Half the volume was used for the whole cell assay and the other half was processed with Clontech x-Tractor kit (Clontech) to obtain the soluble and insoluble fractions of each strain. Both fractions were separated by a 10% SDS-PAGE and visualized with GelCode Blue Stain Reagent (Thermo). |
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Revision as of 04:09, 18 October 2011
• Try another E. coli strain like XL1 Blue, C43, Bl21 SI and others
The 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) thermocycler 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 was first sub-cloned in pGEM T Easy Vector (Promega) and added to the registry (BBa_K633003) and then was cloned to in the plasmid pSB1C3 and added to the registry (BBa_...TBD).
The ompA+sacC construction was generated by joining the biobricks of the araC-PBAD promoter (BBa_I13458 and BBa_K206000), RBS (BBa_B0034), lpp+ompA (BBa_K103006), and sacC (BBa_K633003) with the biobrick standard assembly protocol ( Manual). The expected DNA fragment of the ompA + sacC construct was confirmed by several restriction endonuclease reactions, and used to transform the E.coli strins BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783. The E. coli strains (BL21SI, Rosetta Gami, XL1 Blue, and C43) were obtained from ¿...? and the strain BW27783 was donated by Tec-Monterrey 2010)
The E. coli strains containing the ompA+sacC construct and non-transformed strains as negative controls were cultured in 6 mL of media M9 with glycerol as its unique carbon source. The initial optical density at 600 nm (OD600) was 0.1, from there the batch cultures were incubated at 37°C until the OD600 of 0.6 was attained. The expression was induced with 0.1mM of L-arabinose and the temperature of postinduction was changed to 15 °C. Culture samples collected from the bioreactor were harvested by centrifugation. Half the volume was used for the whole cell assay and the other half was processed with Clontech x-Tractor kit (Clontech) to obtain the soluble and insoluble fractions of each strain. Both fractions were separated by a 10% SDS-PAGE and visualized with GelCode Blue Stain Reagent (Thermo).
