Team:Tec-Monterrey/projectresults/methods

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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/projectresults">methods+results</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/projectresults">results</a></p>
             <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/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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CelD+estA Construction
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<b>1.1. CelD+estA Construction</b>
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CelD+estA Expression
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<b>1.2. CelD+estA Expression</b>
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SacC Amplification
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<b>1.3. CelD+estA Activity</b>
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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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<b>2.1. SacC Amplification</b>
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OmpA+sacC Construction
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<b>2.2. OmpA+sacC Construction</b>
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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>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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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.
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OmpA+sacC Expression
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<b>2.3. OmpA+sacC Expression</b>
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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>

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