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Team:CityU HK/project/result tesA
From 2014.igem.org
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<h2 class="sub">Overview :</h2> | <h2 class="sub">Overview :</h2> | ||
| - | <p class="content">We aimed to overexpress the <i>‘tesA</i> gene (which codes for thioesterase I) in <i>E. coli</i> | + | <p class="content">We aimed to overexpress the <i>‘tesA</i> gene (which codes for thioesterase I) in <i>E. coli</i> to (1) enhance the conversion of fatty acyl-CoA to free fatty acid, and (2) bypass the β-oxidation pathway for fatty acid metabolism. To facilitate this strategy, we have subcloned the <i>‘tesA</i> coding sequence downstream of the PBAD promoter in the pSB1C3 vector. <i>E. coli</i> TOP10 cells (Invitrogen) was used as the host to overexpress <i>‘tesA</i>. Expression of the <i>‘tesA</i> gene was measured by quantitative RT-PCR and the effect of ‘TesA overexpression (leading to a possible increase in thioesterase I activity) was indirectly tested by determining the oleic acid concentrations in control and <i>‘tesA</i> recombinant <i>E. coli</i> cells by GC-MS.</p><br><br> |
<h2 class="sub">Results :</h2> | <h2 class="sub">Results :</h2> | ||
| - | <p class="content">Quantitative real-time PCR analyses showed that <i>‘tesA</i> mRNA overexpression was observed only in recombinant <i>E. coli</i> Top10 cells and not in the DH5α host. Expression of <i> | + | <p class="content">Quantitative real-time PCR analyses showed that <i>‘tesA</i> mRNA overexpression was observed only in recombinant <i>E. coli</i> Top10 cells and not in the DH5α host. Expression of ‘<i>tesA</i> was 2.5-fold higher in the recombinant <i>‘tesA E. coli</i> TOP10 cells than the control cells (Figure 1). </p><br> |
<img class="displayed" src="https://static.igem.org/mediawiki/2014/b/b3/CityU_HK_project_result_tesA1.png" width="35%"><br> | <img class="displayed" src="https://static.igem.org/mediawiki/2014/b/b3/CityU_HK_project_result_tesA1.png" width="35%"><br> | ||
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<th>Figure 1. </th> | <th>Figure 1. </th> | ||
| - | <td><b>Quantitative RT-PCR of <i>‘tesA</i> expression in <i>E. coli</i> DH5α and TOP10 host cells.</b> Expression of <i>‘tesA</i> gene was determined in control and <i>‘tesA</i> recombinant <i>E.coli</i> DH5α and <i>E. coli</i> TOP10 cells. <i>E. coli</i> cells were cultured overnight in LB + oleic acid ( | + | <td><b>Quantitative RT-PCR of <i>‘tesA</i> expression in <i>E. coli</i> DH5α and TOP10 host cells.</b> Expression of the <i>‘tesA</i> gene was determined in control and <i>‘tesA</i> recombinant <i>E.coli</i> DH5α and <i>E. coli</i> TOP10 cells. <i>E. coli</i> cells were cultured overnight in LB + oleic acid (3.5 mM) medium. Cells were harvested for total RNA extraction and lipid extraction for GC-MS analysis. </td> |
</tr> | </tr> | ||
</table><br> | </table><br> | ||
| - | <p class="content">For the fatty acid analysis in <i>E. coli</i> TOP10 cells, the levels of both oleic acid and | + | <p class="content">For the fatty acid analysis in <i>E. coli</i> TOP10 cells, the levels of both oleic acid and palmitic acid were elevated in recombinant <i>‘tesA</i> TOP10 cells (but not recombinant DH5α cells) as compared to the control host cells (Figures 2A and 2B). The results suggested that the increase in oleic acid and palmitic acid levels in the recombinant TOP10 cells may be due to increased expression of the <i>‘tesA</i> gene (and hence thioesterase I activity) in these <i>E. coli</i> cells. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.</p> |
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<tr> | <tr> | ||
<th>Figure 2. </th> | <th>Figure 2. </th> | ||
| - | <td><b>Intracellular levels of oleic acid and palmitic acid in recombinant <i>‘tesA</i> and control <i>E. coli</i> cells.</b> (A). Oleic acid concentration in control DH5α / TOP10 cells (<font color=#FFC000>Yellow</font>), and <i>‘tesA</i> recombinant DH5α /TOP10 cells (<font color=#A5A5A5>Gray</font>). (B). Palmitic acid concentration in control DH5α / TOP10 cells (<font color=#FFC000>Yellow</font>), and <i>‘tesA</i> recombinant DH5α /TOP10 cells (<font color=#A5A5A5>Gray</font>). </td> | + | <td><b>Intracellular levels of oleic acid and palmitic acid in recombinant <i>‘tesA</i> and control <i>E. coli</i> cells.</b> (A). Oleic acid concentration in control DH5α / TOP10 cells (<font color=#FFC000>Yellow</font>), and <i>‘tesA</i> recombinant DH5α /TOP10 cells (<font color=#A5A5A5>Gray</font>). (B). Palmitic acid concentration in the control DH5α / TOP10 cells (<font color=#FFC000>Yellow</font>), and <i>‘tesA</i> recombinant DH5α /TOP10 cells (<font color=#A5A5A5>Gray</font>). </td> |
</tr> | </tr> | ||
Revision as of 13:22, 16 October 2014
Overexpression of ‘tesA Gene and Effect on Conversion of Fatty acyl-CoA to Fatty acid
Overview :
We aimed to overexpress the ‘tesA gene (which codes for thioesterase I) in E. coli to (1) enhance the conversion of fatty acyl-CoA to free fatty acid, and (2) bypass the β-oxidation pathway for fatty acid metabolism. To facilitate this strategy, we have subcloned the ‘tesA coding sequence downstream of the PBAD promoter in the pSB1C3 vector. E. coli TOP10 cells (Invitrogen) was used as the host to overexpress ‘tesA. Expression of the ‘tesA gene was measured by quantitative RT-PCR and the effect of ‘TesA overexpression (leading to a possible increase in thioesterase I activity) was indirectly tested by determining the oleic acid concentrations in control and ‘tesA recombinant E. coli cells by GC-MS.
Results :
Quantitative real-time PCR analyses showed that ‘tesA mRNA overexpression was observed only in recombinant E. coli Top10 cells and not in the DH5α host. Expression of ‘tesA was 2.5-fold higher in the recombinant ‘tesA E. coli TOP10 cells than the control cells (Figure 1).
| Figure 1. | Quantitative RT-PCR of ‘tesA expression in E. coli DH5α and TOP10 host cells. Expression of the ‘tesA gene was determined in control and ‘tesA recombinant E.coli DH5α and E. coli TOP10 cells. E. coli cells were cultured overnight in LB + oleic acid (3.5 mM) medium. Cells were harvested for total RNA extraction and lipid extraction for GC-MS analysis. |
|---|
For the fatty acid analysis in E. coli TOP10 cells, the levels of both oleic acid and palmitic acid were elevated in recombinant ‘tesA TOP10 cells (but not recombinant DH5α cells) as compared to the control host cells (Figures 2A and 2B). The results suggested that the increase in oleic acid and palmitic acid levels in the recombinant TOP10 cells may be due to increased expression of the ‘tesA gene (and hence thioesterase I activity) in these E. coli cells. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.
| Figure 2. | Intracellular levels of oleic acid and palmitic acid in recombinant ‘tesA and control E. coli cells. (A). Oleic acid concentration in control DH5α / TOP10 cells (Yellow), and ‘tesA recombinant DH5α /TOP10 cells (Gray). (B). Palmitic acid concentration in the control DH5α / TOP10 cells (Yellow), and ‘tesA recombinant DH5α /TOP10 cells (Gray). |
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