Team:CSU Fort Collins/Notebook/Protocols=Gel

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       <ul>
       <ul>
         <li><a href='/Team:CSU_Fort_Collins/Members/'><span>Members</span></a></li>
         <li><a href='/Team:CSU_Fort_Collins/Members/'><span>Members</span></a></li>
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         <li class='last'><a href='/Team:CSU_Fort_Collins/Sponsors/'><span>Sponsors</span></a></li>
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        <li><a href='/Team:CSU_Fort_Collins/Sponsors/'><span>Sponsors</span></a></li>
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         <li class='last'><a href='/Team:CSU_Fort_Collins/Acknowledgements/'><span>Acknowledgements</span></a></li>
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       </ul>
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       <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/DailyNotes'><span>Daily Notes</span></a>
       <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/DailyNotes'><span>Daily Notes</span></a>
         <ul>
         <ul>
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         <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/'><span>Biosensor</span></a>
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         <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/Jun'><span>Biosensor</span></a>
             <ul>
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               <li><a href="#"><span>June</span></a></li>
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               <li><a href="/Team:CSU_Fort_Collins/Notebook/Biosensor/Jun"><span>June</span></a></li>
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               <li><a href="#"><span>July</span></a></li>
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               <li><a href="/Team:CSU_Fort_Collins/Notebook/Biosensor/Jul"><span>July</span></a></li>
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              <li><a href='#'><span>August</span></a></li>
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               <li class='last'><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/Aug'><span>August</span></a></li>
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               <li class='last'><a href="#"><span>September</span></a></li>
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             </ul>
             </ul>
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         </li>
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         <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/'><span>Breakdown</span></a>
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         <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul'><span>Breakdown</span></a>
             <ul>
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               <li><a href="/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul"><span>July</span></a></li>
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               <li><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Aug'><span>August</span></a></li>
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               <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/Breakdown/Sep"><span>September</span></a></li>
             </ul>
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         <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/HVP/'><span>High-Value Product</span></a>
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         <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/HVP/Jun'><span>High-Value Product</span></a>
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               <li><a href="/Team:CSU_Fort_Collins/Notebook/HVP/Jun"><span>June</span></a></li>
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              <li><a href="#"><span>July</span></a></li>
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               <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/HVP/Jul"><span>July</span></a></li>
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             </ul>
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         </li>
         </li>
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         <li li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/KillSwitch/'><span>Kill Switch</span></a>
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         <li li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/KillSwitch/Jul'><span>Kill Switch</span></a>
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               <li><a href="#"><span>July</span></a></li>
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               <li><a href="/Team:CSU_Fort_Collins/Notebook/KillSwitch/Jul"><span>July</span></a></li>
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               <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/KillSwitch/Sep"><span>September</span></a></li>
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   </ul>
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   <li class='has-sub'><a href='/Team:CSU_Fort_Collins/HumPrac/'><span>Human Practices</span></a>
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         <li><a href='/Team:CSU_Fort_Collins/Collab/'><span>Collaboration</span></a></li>
         <li><a href='/Team:CSU_Fort_Collins/Collab/'><span>Collaboration</span></a></li>
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   <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Parts/'><span>Achievements</span></a>
       <ul>
       <ul>
         <li><a href='/Team:CSU_Fort_Collins/Parts/'><span>Parts</span></a></li>
         <li><a href='/Team:CSU_Fort_Collins/Parts/'><span>Parts</span></a></li>
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   <li><a href='/Team:CSU_Fort_Collins/Safety/'><span>Safety</span></a></li>
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   <center><h1>Gel Electrophoresis Protocol</h1>
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    <h1>Welcome to CSU Fort Collin's 2014 Wiki</h1>
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      <h3>Our Project</h3>
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      Approximately 3 billion gallons per year of used frying oil are produced in the U.S. alone. While some recycling efforts are put forth to turn used frying oil into biodiesel and some companies are working on more effective ways to recycle the frying oil, the iGEM team at Colorado State University is working to turn spent frying oil into a high value product. There are four major components to this year’s project including; the breakdown of frying oil, a biosensor for detecting the breakdown of frying oil, production of a high value product, and a kill switch to kill the bacteria if they were to be released into the environment. Our team aims to put each of these components into Escherichia coli.  
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In order to detect that the process is working we have developed a biosensor that will detect the breakdown of Acyl-CoA, a byproduct of the breakdown pathway of lipids, in E. coli. Our biosensor is being constructed by inserting a novel promoter in front of DNA expressing a Green Fluorescent Protein (GFP). This promoter is activated by Acyl-CoA, a byproduct of the breakdown pathway of lipids in E. coli. This will result in E. coli expressing GFP when successful breakdown is occurring.
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      <a href='#' id='ftoc'>&#8592; Full Table of Contents</a><br>
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In order to break down the frying oil we will be taking advantage of the cell’s natural ability to break down fatty acids for use in the Kreb’s cycle. We will be upregulating the limiting enzymes that aid in the breakdown of fatty acids in order to have the cell produce more Acetyl-CoA.
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        <a href='#'>Gibson Assembly</a>
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This Acetyl-CoA can then be used in the mevalonate pathway. This pathway is common to most plants as well as yeast. We will be taking the genes from yeast and constructing two operons in order to produce isopentenyl pyrophosphate (IPP). While both IPP and dimethylallyl pyrophosphate (DMAPP) are both naturally produced in E. coli  through the non-mevalonate pathway , the mevalonate pathway provides a more efficient route from acetyl-CoA to terpenoid production than the alternative. Our high-value product will be a terpenoid that has yet to be determined.
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For our kill switch, we will be using a gene named Killer Red which is a mutant of hydrozoan chromoprotein anm2CP from Anthomedusae sp. DC-2005 . Our team plans to insert this gene into our construct and by doing so, it will provide a fail-safe in the unlikely event of our E. coli escaping into the environment. The Killer Red gene has a repressible promoter in which tryptophan acts as the repressor. There will be tryptophan present while the cell is used to break down the frying oil, but is not highly present in the environment. Without the presence of tryptophan the Killer Red gene will be activated and white light will produce reactive oxygen species within the cell, causing cell death.
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        <a href='#'>Cloning Genes</a>
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Our biosensor is being constructed by inserting a novel promoter in front of DNA expressing a Green Fluorescent Protein (GFP). This promoter is activated by Acyl-CoA, a byproduct of the breakdown pathway of lipids in E. coli. This will result in E. coli expressing GFP when successful breakdown is occurring.
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        <a href='#'>Plasmid Miniprep</a>
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        <a href='#'>Yeast DNA Extraction</a>
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        <a href='#'>Gel Electrophoresis</a>
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        <a href='#'>PCR Product Purification</a>
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    <center><table><center><td><p>© Colorado State University iGEM 2014 | Colorado State University, Campus Delivery 1370, Fort Collins, CO 80523-1370 | <a href="/Team:CSU_Fort_Collins/Contact/" > Contact Us </a>| <a href="/Team:CSU_Fort_Collins/Sponsors/"> Become a Sponsor </a> | <a href="http://www.facebook.com/csu.igem.2014" >Facebook</a> | <a href="http://twitter.com/CSU_iGEM" >Twitter</a></td></center></p>
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          <li>Using a balance, mass out 1.0 gram of agarose. Mix this with 100 mL of TAE 1X Buffer.</li>
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          <li>Microwave the mixture, mixing intermittently, until all the agarose is dissolved and the mixture is homogeneous.</li>
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          <li>Pour the mixture into a gel plate and insert an 8-lane comb. Allow to cool and solidify in the fridge or on the benchtop</li>
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          <li>Mix your samples to run in the gel. Use 10 &#956;L of the DNA Ladder combined with 2 &#956;L SYBR Green in the first lane and for all samples, mix 5 &#956;L of sample with 5 &#956;L of nuclease-free water and 2 &#956;L of SYBR Green/Dye Mixture.</li>
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          <li>Place gel in tray into the gel electrophoresis apparatus; Wells should be located towards the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill.</li>
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          <li>Load 10 &#956;L of each sample into the appropriate wells.</li>
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          <li>Connect wiring and run gel electrophoresis for 1 hour.</li>
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          <li>Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.</li>
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    <center><a href='/Team:CSU_Fort_Collins/Notebook/Protocols=Isolation' id='navi' style='margin-left:-20px; margin-right:40px'> Previous</a> <a href='/Team:CSU_Fort_Collins/Notebook/Protocols=Purify' id='navi'>Next </a></center>
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Latest revision as of 17:50, 17 October 2014

CSU iGEM 2014

Gel Electrophoresis Protocol

Show Table of Contents
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← Full Table of Contents
Gibson Assembly
Cloning Genes
Plasmid Miniprep
Yeast DNA Extraction
Gel Electrophoresis
PCR Product Purification

  1. Using a balance, mass out 1.0 gram of agarose. Mix this with 100 mL of TAE 1X Buffer.
  2. Microwave the mixture, mixing intermittently, until all the agarose is dissolved and the mixture is homogeneous.
  3. Pour the mixture into a gel plate and insert an 8-lane comb. Allow to cool and solidify in the fridge or on the benchtop
  4. Mix your samples to run in the gel. Use 10 μL of the DNA Ladder combined with 2 μL SYBR Green in the first lane and for all samples, mix 5 μL of sample with 5 μL of nuclease-free water and 2 μL of SYBR Green/Dye Mixture.
  5. Place gel in tray into the gel electrophoresis apparatus; Wells should be located towards the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill.
  6. Load 10 μL of each sample into the appropriate wells.
  7. Connect wiring and run gel electrophoresis for 1 hour.
  8. Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.



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