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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<div class="frame" id="frame1" style="background-color:#e5e5e5;">
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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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<center>
<center>
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SacC Amplification
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<b>1.2. CelD+estA Expression</b>
</center>
</center>
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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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<br>
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</p>
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<br>
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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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<br>
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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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<br>
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<br>
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<center>
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<b>2.1. SacC Amplification</b>
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</center>
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<br>
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<p class="textojustif">
<p class="textojustif">
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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 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 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>).
</p>
</p>
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  <br>
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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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<br>
<br>
<p class="textojustif">
<p class="textojustif">
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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> 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 Invitrogen, Novagen, Agilent and ¿?, 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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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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<br>
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<br>
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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>
<br>
<p class="textojustif">  
<p class="textojustif">  
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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 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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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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</p>
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<center>
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<b>2.4. SacC Enzymatic Assays</b>
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</center>
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<br>
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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>

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