Team:CityU HK/project/result

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<h2 class="sub">Experimental Achievements from 15 June – 3 October 2014 :</h2>
<h2 class="sub">Experimental Achievements from 15 June – 3 October 2014 :</h2>
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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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<p class="content">Successfully constructed a Biobrick for a tetracycline regulated fadL-fadD expression system (BBa_K1472606).<br>
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Successfully constructed and characterized a T7-regulated fadL-fadD  system in the pSNAP-tag® (T7)-2 expression vector.<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 FadL and FadD genes in <i>E. coli.</i> FadL codes for a fatty acid transporter protein that moves free fatty acids across the outer cell membrane into the periplasmic space. FadD, 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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Successfully constructed a Biobrick containing a “leaderless” tesA coding sequence (‘tesA) (BBa_K1472601).<br>
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Successfully constructed a translational unit of the ‘tesA gene in pAB1A2. <br>
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Next, <i>E. coli.</i> is engineered to overexpress the TesA 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 acid. 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 L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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Successfully constructed an arabinose-regulated ‘tesA expression system in pSB1C3.<br>
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Successfully constructed a Biobrick containing a Δ15 desaturase gene that is codon-optimized for protein expression in E.coli (BBa_K1472610).<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) converting 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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Successfully constructed a translational unit of a Δ9 desaturase gene in pSB1A2 that is codon-optimized for E. coli.<br>
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Successfully constructed a translational unit of a Δ12 desaturase gene in pSB1A2 that is codon-optimized for E. coli.<br>
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Successfully constructed a translational unit of a Δ15 desaturase gene in pSB1A2 that is codon-optimized for E. coli.<br>
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Successfully constructed a translational unit of a Δ9-Δ12 desaturase gene cluster in pSB1A2.<br>
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Successfully constructed a translational unit of a Δ9-Δ12-Δ15 desaturase gene cluster in pSB1A2.
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Revision as of 08:36, 15 October 2014

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Result Summary

Experimental Achievements from 15 June – 3 October 2014 :

Successfully constructed a Biobrick for a tetracycline regulated fadL-fadD expression system (BBa_K1472606).
Successfully constructed and characterized a T7-regulated fadL-fadD system in the pSNAP-tag® (T7)-2 expression vector.
Successfully constructed a Biobrick containing a “leaderless” tesA coding sequence (‘tesA) (BBa_K1472601).
Successfully constructed a translational unit of the ‘tesA gene in pAB1A2.
Successfully constructed an arabinose-regulated ‘tesA expression system in pSB1C3.
Successfully constructed a Biobrick containing a Δ15 desaturase gene that is codon-optimized for protein expression in E.coli (BBa_K1472610).
Successfully constructed a translational unit of a Δ9 desaturase gene in pSB1A2 that is codon-optimized for E. coli.
Successfully constructed a translational unit of a Δ12 desaturase gene in pSB1A2 that is codon-optimized for E. coli.
Successfully constructed a translational unit of a Δ15 desaturase gene in pSB1A2 that is codon-optimized for E. coli.
Successfully constructed a translational unit of a Δ9-Δ12 desaturase gene cluster in pSB1A2.
Successfully constructed a translational unit of a Δ9-Δ12-Δ15 desaturase gene cluster in pSB1A2.



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