Team:Tec-Monterrey/projectresults/methods

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     <div class="panelcontent" style="">
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        <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectoverview">overview</a></p>
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                  <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectoverview">overview</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectparts">parts</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectparts">parts</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectmodeling">genetic frame</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectmodeling">genetic frame</a></p>
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             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/safetypage">safety</a></p>
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             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectresults/methods">methods</a></p>
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            <p><a href="https://2011.igem.org/Team:Tec-Monterrey/teamha">human practice</a></p>
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            <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectnotebook">notebook</a></p>
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             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectresults">results</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectresults">results</a></p>
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            <p><a href="https://2011.igem.org/Team:Tec-Monterrey/teamha">human approach</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectprotocols">protocols</a><p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectprotocols">protocols</a><p>
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            <p><a href="https://2011.igem.org/Team:Tec-Monterrey/safetypage">safety</a></p>
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            <p><a href="https://2011.igem.org/Team:Tec-Monterrey/projectnotebook">notebook</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/sampledata">sample data</a></p>
             <p><a href="https://2011.igem.org/Team:Tec-Monterrey/sampledata">sample data</a></p>
           </div>
           </div>
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• Try another E. coli strain like XL1 Blue, C43, Bl21 SI and others<br>
 
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<center>
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<b>1.1. CelD+estA Construction</b>
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</center>
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<br>
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<p class="textojustif">
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The celD+estA 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+phoA signal peptide+celD (<a href="http://partsregistry.org/Part:BBa_K633002">BBa_K633002</a>) and linker+estA (<a href="http://partsregistry.org/Part:BBa_K633001">BBa_K633001</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 celD+estA construct was confirmed by several restriction endonuclease reactions, and used to transform the <i>Escherichia coli</i> strains BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783. The <i>E. coli</i> strains BL21SI, Rosetta Gami, XL1 Blue, and C43 were obtained from Invitrogen, Novagen, Agilent and Lucigen, respectively, and the strain BW27783 was donated by <a href="https://2010.igem.org/Team:Tec-Monterrey">Tec-Monterrey 2010</a>.
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SacC Amplification
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<b>1.2. CelD+estA Expression</b>
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<p class="textojustif">
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The <i>E. coli</i> strains containing the celD+estA construct and non-transformed strains as negative controls were cultured in 6 mL of LB Miller Broth. 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 an 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 30 °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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</p>
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<center>
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<b>1.3. CelD+estA Activity</b>
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</center>
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<p class="textojustif">
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The IUPAC Filter Paper Assay was used to determine the celD+estA activity.  The <i>E. coli</i> strain , Rosetta Gami, was used as a host for the expression of the chimeric protein because it has an improved protein folding system. The assay was applied to the whole-cells, but these were also lysated with x-Tractor Cell lysis Buffer (Clontech), which separated them into soluble and insoluble fractions. The negative controls (C-) of all the samples were non-transformed cells. In the whole-cell cellulase activity experiment and in the cellulase activity of cell-lysates experiment, a t-test was done  with an alpha of 0.05 to prove the hypothesis.
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<center>
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<b>2.1. SacC Amplification</b>
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</center>
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<p class="textojustif">
<p class="textojustif">
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Sac C 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>, ¿? mM of forward and reverse primers. PCR was performed in an ¿? (¿?) using the following program: ¿? C for ¿? min, 30 cycles of ¿? C for ¿? s, 56.4 C for ¿? s, and 72 C for ¿? min, and finally an extension step at 72 C for ¿? 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_ ¿?).
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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 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>).
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<center>
<center>
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OmpA+sacC Construction
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<b>2.2. OmpA+sacC Construction</b>
</center>
</center>
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<p class="textojustif">
<p class="textojustif">
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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 (¿cita?). The expected DNA fragment size (XX bp) 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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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> strains BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783.
  </p>
  </p>
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<center>
<center>
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OmpA+sacC Expression
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<b>2.3. OmpA+sacC Expression</b>
</center>
</center>
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<br>
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<p class="textojustif">  
<p class="textojustif">  
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The <i>E. coli</i> 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 (OD<sub>600</sub>) of 0.1. The batch cultures were performed at 37C until the OD<sub>600</sub> 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 X % (w/v) Coomassie Brilliant Blue R250.
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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 an 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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<b>2.4. SacC Enzymatic Assays</b>
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<p class="textojustif">
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To determine the ompA+sacC activity, EnzyChromTM Fructose Assay Kit (kindly donated by PhD. Fernández) was used. The <i>E. coli</i> BL21 SI was used for the expression of the fusion protein. The assay was applied to the whole-cells, and non-transformed cells were used as a negative control. A t-test was done with an alpha value of 0.05 to compare fructose concentrations of each sample.
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</p>

Latest revision as of 20:51, 20 October 2011

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