Difference between revisions of "Team:MSU-Michigan/Design"

 
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<h1>Design</h1>
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:MSU-Michigan/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
 
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<img style="-webkit-user-select: none;background-position: 0px 0px, 10px 10px;background-size: 20px 20px;background-image:linear-gradient(45deg, #eee 25%, transparent 25%, transparent 75%, #eee 75%, #eee 100%),linear-gradient(45deg, #eee 25%, white 25%, white 75%, #eee 75%, #eee 100%);cursor: zoom-in;" src="https://static.igem.org/mediawiki/2017/0/05/MSU-Michigan_bioreactorassembly.png" width="800" height="403">
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<p> Process for simple bioreactor assembly used to measure current produced by <i>Shewanella Oneidensis</i> strains when connected to a potentiostat </p>
 
<p> Process for simple bioreactor assembly used to measure current produced by <i>Shewanella Oneidensis</i> strains when connected to a potentiostat </p>
  
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<h5>What should this page contain?</h5>
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<ul>
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<li>Explanation of the engineering principles your team used in your design</li>
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<li>Discussion of the design iterations your team went through</li>
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<li>Experimental plan to test your designs</li>
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Single chambered bioreactors were designed to be economical and functional.  Reactors are run on magnetic stir plates and connected to a lab grade potentiostat.
  
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<h5>Inspiration</h5>
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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<h2>Plasmid</h2>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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    To produce an electric current from Shewanella oneidensis MR-1 in response to chemicals in a sample, the mtrB gene was first removed from the genome (ΔmtrB) then re-introduced to the bacteria in a plasmid vector under an inducible promoter. The first promoter chosen was the well-studied IPTG inducible T7A1 promoter. Next, nine promoter sequences shown to respond to different effector molecules were identified and amplified from the S. oneidensis MR-1 genome with the addition of the restriction enzyme cut sites for AatII and NdeI on either end.
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Double digestion of both the vector and promoters using AatII and NdeI removed the T7A1 promoter and produced compatible sticky ends in the new promoter for insertion via ligation.
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The designed constructs were individually transformed into E. coli WM3064, sequenced for confirmation, then conjugated into S. oneidensis MR-1 ∆mtrB. The S. oneidensis were then tested for induction through quantification of GFP fluorescence (marker protein) and current production using a spectrophotometer and bioreactor, respectively.
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Latest revision as of 02:56, 2 November 2017

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Design

Process for simple bioreactor assembly used to measure current produced by Shewanella Oneidensis strains when connected to a potentiostat

Bioreactors

Single chambered bioreactors were designed to be economical and functional. Reactors are run on magnetic stir plates and connected to a lab grade potentiostat.

Plasmid

To produce an electric current from Shewanella oneidensis MR-1 in response to chemicals in a sample, the mtrB gene was first removed from the genome (ΔmtrB) then re-introduced to the bacteria in a plasmid vector under an inducible promoter. The first promoter chosen was the well-studied IPTG inducible T7A1 promoter. Next, nine promoter sequences shown to respond to different effector molecules were identified and amplified from the S. oneidensis MR-1 genome with the addition of the restriction enzyme cut sites for AatII and NdeI on either end. Double digestion of both the vector and promoters using AatII and NdeI removed the T7A1 promoter and produced compatible sticky ends in the new promoter for insertion via ligation.

The designed constructs were individually transformed into E. coli WM3064, sequenced for confirmation, then conjugated into S. oneidensis MR-1 ∆mtrB. The S. oneidensis were then tested for induction through quantification of GFP fluorescence (marker protein) and current production using a spectrophotometer and bioreactor, respectively.

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