Team:Manaus Amazonas-Brazil/Overview

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<html><head><title>overview.docx</title><meta content="text/html; charset=UTF-8" http-equiv="content-type"><style type="text/css">@import url('https://themes.googleusercontent.com/fonts/css?kit=wAPX1HepqA24RkYW1AuHYA');ol{margin:0;padding:0}.c0{color:#ffffff;font-size:12pt;font-style:italic;font-family:"Times New Roman"}.c8{max-width:425.2pt;background-color:#ffffff;padding:70.8pt 85pt 70.8pt 85pt}.c2{height:11pt;text-align:justify;direction:ltr}.c5{text-align:justify;direction:ltr}.c1{color:#ffffff;font-size:12pt}.c6{height:11pt;direction:ltr}.c9{text-align:center;direction:ltr}.c11{color:#ffffff;font-size:10pt}.c4{font-family:"Times New Roman";font-weight:bold}.c7{font-family:"Times New Roman"}.c3{font-family:"Britannic Bold"}.title{padding-top:24pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:36pt;font-family:"Calibri";font-weight:bold;padding-bottom:6pt}.subtitle{padding-top:18pt;line-height:1.1500000000000001;text-align:left;color:#666666;font-style:italic;font-size:24pt;font-family:"Georgia";padding-bottom:4pt}li{color:#000000;font-size:11pt;font-family:"Calibri"}p{color:#000000;font-size:11pt;margin:0;font-family:"Calibri"}h1{padding-top:24pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:24pt;font-family:"Calibri";font-weight:bold;padding-bottom:6pt}h2{padding-top:18pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:18pt;font-family:"Calibri";font-weight:bold;padding-bottom:4pt}h3{padding-top:14pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:14pt;font-family:"Calibri";font-weight:bold;padding-bottom:4pt}h4{padding-top:12pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:12pt;font-family:"Calibri";font-weight:bold;padding-bottom:2pt}h5{padding-top:11pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:11pt;font-family:"Calibri";font-weight:bold;padding-bottom:2pt}h6{padding-top:10pt;line-height:1.1500000000000001;text-align:left;color:#000000;font-size:10pt;font-family:"Calibri";font-weight:bold;padding-bottom:2pt}</style></head><body class="c8"><br><br><br><br><br><br><br><br><p class="c9"><span class="c1 c3">Overview</span></p><p class="c2"><span class="c1"></span></p><p class="c5"><span class="c1 c7">We want a bacteria efficient in metabolize fatty acid and liberate electrons to the media. For this we have THREE approaches:</span></p><p class="c5"><span class="c1 c4">1&deg; Avoid the FadR (protein that prevents the break down of fatty acid) expression</span></p><p class="c5"><span class="c1 c7">We know </span><span class="c0">Shewanella </span><span class="c1 c7">doesn&rsquo;t metabolize fatty acid all the time, mainly due to the media composition. If it doesn&rsquo;t have fatty acid, the bacteria won&rsquo;t produce the enzymes that degrade the fatty acid. But if you have fatty acid, then yes, enzymes that break it will be produced. So, to not produce such enzymes, regulatory proteins, such as protein FadR inhibitory enter in scene. FadR is a protein that inhibits the metabolism of fatty acid, she connects on the operator preventing the RNA polymerase to continue and transcribe the information to produce proteins that bacteria need.</span></p><p class="c5"><span class="c1 c7">How are we going to avoid de FadR expression?</span></p><p class="c5"><span class="c1 c7">Forward and Reverse primers were constructed for parts of the FadR gene, a 5&rsquo; and 3&rsquo; pieces were amplified (up and down) in order to design a construction in this following structure: gene up + tetracycline resistance gene + gene down. </span></p><p class="c5"><span class="c1 c7">So the wild, normal gene leaves and the construction takes place by recombination. </span></p><p class="c2"><span class="c1"></span></p><p class="c5"><span class="c1 c4">2&deg; Increase the FadL expression.</span></p><p class="c5"><span class="c1 c7">In </span><span class="c0">Shewanella putrefaciens</span><span class="c1 c7">, there is a protein that works in the intaking fatty acid called FadL. The FadL gene we introduced begin to overexpress this proteins, enabling that more fatty acids enter inside the cell and the generation of ATP and electrons increases.</span></p><p class="c5"><span class="c1 c4">3&deg; Increase the FadD expression.</span></p><p class="c5"><a name="h.gjdgxs"></a><span class="c1 c7">The fatty acid enter in the bacteria trough FadL protein, soon, it is gonna be catalized for a serie of proteins on bacteria intern membrane. One of this proteins is the FadD that works ligating CoA in fatty acid chain </span><span class="c1 c7 c10">preparing it to get into the Beta-Oxidation metabolic pathway.</span><span class="c10 c7 c11">&nbsp;</span><span class="c1 c7">&nbsp;The FadD gene were introduced in a vector and transformated &nbsp;in </span><span class="c0">S. putrefaciens</span><span class="c1 c7">. She will begin to overexpress these proteins, signaling and enabling that more fatty acids enter inside the cell to be degradated.</span></p><p class="c2"><span class="c1"></span></p><p class="c2"><span class="c1"></span></p><p class="c2"><span class="c1"></span></p><p class="c6"><span class="c1"></span></p></body></html>
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<z1 >Overview</z1><br>
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  <br><br>
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Our project aim increased the degradation of fatty acid (Beta
 +
 
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oxidation pathway) in Shewanella putrefaciens. So, we choose 2
 +
 
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methodologies to do that.
 +
 
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1° inhibition of the FadR expression (fatty acid  
 +
 
 +
metabolism regulator protein)
 +
 
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The beta-oxidation is not a constituve pathway but with the  
 +
 
 +
mutation of the transcription factor, FadR, which functions as a  
 +
 
 +
switch that co-ordinately regulates the machinery required for fatty  
 +
 
 +
acid beta-oxidation, we hope turn this pathway constituve. To
 +
 
 +
increased the degradation of fatty acid by S. Putrefaciens.
 +
 
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2° Increase the FadL and FadD expression.
 +
 
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Our second step in  genetic engineering of  Shewanella is  
 +
 
 +
overexpression of two important genes for beta oxidation : (1)
 +
 
 +
The FadL, Long-chain fatty acid outer membrane transporte, is a
 +
 
 +
gene of fatty acid transport protein, outer membrane; long-chain
 +
 
 +
fatty acid receptor to enhance the long chain fatty acids intake
 +
 
 +
by Shewanella putrefaciens and (2) The FadD, Long-chain-fattyacid-CoA ligase, ligates CoA in fatty acid chain preparing it to get  
 +
 
 +
into the Beta-Oxidation metabolic pathway. These overexpression
 +
 
 +
will increased B-oxidation and therefore ATP, H+ and electrons
 +
 
 +
production.
 +
 
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Revision as of 02:52, 28 September 2013





Overview




Our project aim increased the degradation of fatty acid (Beta oxidation pathway) in Shewanella putrefaciens. So, we choose 2 methodologies to do that. 1° inhibition of the FadR expression (fatty acid metabolism regulator protein) The beta-oxidation is not a constituve pathway but with the mutation of the transcription factor, FadR, which functions as a switch that co-ordinately regulates the machinery required for fatty acid beta-oxidation, we hope turn this pathway constituve. To increased the degradation of fatty acid by S. Putrefaciens. 2° Increase the FadL and FadD expression. Our second step in genetic engineering of Shewanella is overexpression of two important genes for beta oxidation : (1) The FadL, Long-chain fatty acid outer membrane transporte, is a gene of fatty acid transport protein, outer membrane; long-chain fatty acid receptor to enhance the long chain fatty acids intake by Shewanella putrefaciens and (2) The FadD, Long-chain-fattyacid-CoA ligase, ligates CoA in fatty acid chain preparing it to get into the Beta-Oxidation metabolic pathway. These overexpression will increased B-oxidation and therefore ATP, H+ and electrons production.




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