Team:CityU HK/project/futureplan

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     <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>
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     <td><b>Replication of quantitative RT-PCR and GC-MS experiments.</b><br>
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Due to time constraint, the RT-PCR and GC-MS experiments on the different recombinant E. coli clones were performed only once, and will have to be repeated in future studies in order to verify the statistical significance of the data.
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    <td><b>Construction of Δ9-Δ12-Δ15 desaturase gene cluster downstream of the LacI promoter in pSB1C3. </b><br>
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For overexpression studies of the Δ9-Δ12-Δ15 desaturase gene cluster in E. coli, we will repeat subcloning of the  desaturase gene cluster into pSB1C3 to create a regulatable expression system for ALA production.
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    <td><b>Cotransformation of TOP10 cells with three different gene cassettes. </b><br>
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Attempts will be made to co-transform E. coli TOP10 cells with (1) the fadL-fadD expression cassette, (2) the ‘tesA expression cassette, and (3) the Δ9-Δ12-Δ15 desaturase gene cassette to construct a Fit Coli strain. Expression of the gene cassettes will be analysed by qRT-PCR, whilst fatty acid uptake and ALA production will be monitored by GC-MS analyses.
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    <td><b>Studies on efficiency of ALA exportation by Fit Coli strain.  </b><br>
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Because unsaturated fatty acids are expensive sources of carbon and energy from the perspective of a cell, and an ALA export protein may not be present in E. coli to facilitate ALA export for human uptake, we will consider a pH-induced autolysis system to facilitate extracellular ALA release. When bacteria reach a certain section of the gut of a particular pH, the cell will open up so the human gut may gain access to the useful ALA. Alternatively, a bile salt-inducible system to activate yet another engineered gene from E. coli, which encodes the MazF protein (a stable toxin) that initiates program death in the cells will also be tested (http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04956.x/pdf).
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<p class="content">Obesity is a worldwide problem that contributes to a variety of human diseases such as diabetes and cancer, as well as psychological problems such as eating disorder and anxiety. Traditional remedies such as dieting or over-exercising can be ineffective and often lead to depression, given that both means could lead to obvious physical and mental unpleasantness. Obesity, in many cases, is linked to the accumulation of excessive free fatty acids from food. Therefore removing these fatty acids from the system could be the key to combating obesity. In an attempt to alleviating the obesity problem without causing adverse impact on one’s quality of life, we genetically engineered an <i>Escherichia coli</i> strain to confer upon it the capacity to both remove excess fat from our diet and convert it into a useful metabolic intermediate, linolenic acid (ALA). We named this genetically modified bacterium, “Fit Coli”.<br><br>
 
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We designed Fit Coli to do the job in several steps. The first step involves increasing its ability to take up excess free fatty acids from the external environment (e.g. human gut) which, theoretically, can be achieved by overexpressing the <i>fadL</i> and <i>fadD</i> genes in <i>E. coli</i>. <i>fadL</i> codes for a fatty acid transporter protein that moves free fatty acids across the outer cell membrane into the periplasmic space. <i>fadD</i>, on the other hand, codes for a fatty acyl-CoA synthetase which adds a coenzyme A (CoA) moiety to fatty acids and then transports the resulting fatty acyl-CoA across the inner membrane into the cytosol.  <br><br>
 
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Next, <i>E. coli.</i> is engineered to overexpress the <i>tesA</i> gene that codes for an acyl-CoA thioesterase which removes the CoA moiety from fatty acyl-CoA molecules, thereby restoring the molecule back to the form of free fatty acids. This strategy is aimed at diverting fatty acids from the beta-oxidation pathway which degrades fatty acids into acetyl-CoA to produce ATP. Lastly, the free fatty acids are converted into ALA under the catalysis of the three engineered enzymes - ∆9 desaturase, ∆12 desaturase and ∆15 desaturase – in the Fit Coli. <br><br>
 
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In short, Fit Coli is designed to combat obesity by its ability (1) to take up various excess C18 fatty acids such as stearic acid, oleic acid and linoleic acid present in our foods and (2) to convert them into ALA which can then be used by the human body to make docosahexaenoic acid/eicosanoids that are beneficial to humans.<br><br> </p>
 
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Revision as of 07:49, 17 October 2014

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Future plan

Replication of quantitative RT-PCR and GC-MS experiments.
Due to time constraint, the RT-PCR and GC-MS experiments on the different recombinant E. coli clones were performed only once, and will have to be repeated in future studies in order to verify the statistical significance of the data.
Construction of Δ9-Δ12-Δ15 desaturase gene cluster downstream of the LacI promoter in pSB1C3.
For overexpression studies of the Δ9-Δ12-Δ15 desaturase gene cluster in E. coli, we will repeat subcloning of the desaturase gene cluster into pSB1C3 to create a regulatable expression system for ALA production.
Cotransformation of TOP10 cells with three different gene cassettes.
Attempts will be made to co-transform E. coli TOP10 cells with (1) the fadL-fadD expression cassette, (2) the ‘tesA expression cassette, and (3) the Δ9-Δ12-Δ15 desaturase gene cassette to construct a Fit Coli strain. Expression of the gene cassettes will be analysed by qRT-PCR, whilst fatty acid uptake and ALA production will be monitored by GC-MS analyses.
Studies on efficiency of ALA exportation by Fit Coli strain.
Because unsaturated fatty acids are expensive sources of carbon and energy from the perspective of a cell, and an ALA export protein may not be present in E. coli to facilitate ALA export for human uptake, we will consider a pH-induced autolysis system to facilitate extracellular ALA release. When bacteria reach a certain section of the gut of a particular pH, the cell will open up so the human gut may gain access to the useful ALA. Alternatively, a bile salt-inducible system to activate yet another engineered gene from E. coli, which encodes the MazF protein (a stable toxin) that initiates program death in the cells will also be tested (http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04956.x/pdf).


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