Difference between revisions of "Team:Dalhousie/Experiments"

 
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       <li ><a href="https://2017.igem.org/Team:Dalhousie/test3" style=" color: white;">Home</a></li>
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           <li ><a href="https://2017.igem.org/Team:Dalhousie/Description" style=" color: white;" >Description</a></li>
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          <li class="active"><a href="https://2017.igem.org/Team:Dalhousie/Experiments"style=" color: white;">Experiments</a></li>
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                         <a href="mailto:igemdalhousie@gmail.com" ><i class="fa fa-envelope-o fa-fw" style=" color: rgba(149, 48, 41, 1);"></i> <span class="network-name"></span></a>
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                         <a href="mailto:igemdalhousie@gmail.com" ><i class="fa fa-envelope-o fa-fw" style=" color: white;" ></i> <span class="network-name"></span></a>
 
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                         <a target="_blank" href="https://twitter.com/dalhousie_igem?lang=en" ><i class="fa fa-twitter fa-fw" style=" color: rgba(61, 145, 229, 1);"></i> <span class="network-name"></span></a>
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<img src= "https://static.igem.org/mediawiki/2016/a/a9/T--Dalhousie_Halifax_NS--CBForest.jpeg" style="top:0px; left:0px; padding-bottom:0px; position:fixed; width:100%; height: 100%; z-index:-100; " align="center" height="70%" width="70%">
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<img src= "https://static.igem.org/mediawiki/2016/9/9b/T--Dalhousie_Halifax_NS--experiments.jpeg" style="top:0px; left:0px; padding-bottom:0px; position:fixed; width:100%; height: 100%; z-index:-100; " align="center" height="70%" width="70%">
  
  
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</br>Project Description</br>
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WHY</br>
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As fossil fuels continue to run out across the globe, many people are looking to alternate sources of energy that are renewable and eco-friendlier. One of these options is biofuel which is fuel made from organic matter. Most commonly made from ethanol, biofuel or bioethanol can be used as a fuel for vehicles in its’ pure form. In the field of biofuel production, bioethanol made from cellulose continues to be the dominant form. However harsh methods are required to be able to extract the sugars from cellulose and convert it to ethanol.
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Atlantic Canada’s main export is wood, pulp, and paper. Many of us on the team grew up next to pulp and paper mills and saw the hazardous waste expelled by the processes in these buildings. Extraction of usable materials from wood is fairly inefficient, leaving behind wood waste that could be used for biofuel production if broken down and converted to ethanol. The only question is: how?</br>
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WHAT</br>
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The Dalhousie iGEM team this year is focused on using the microbiome of the porcupine to solve this conversion of wood waste to ethanol. A huge part of the porcupine’s diet is made from bark. Unable to digest the cellulose, hemi-cellulose, and lignin in the bark, the gut bacteria of the porcupine do the work instead. We hypothesized that the microbiome of the porcupine would contain enzymes that convert cellulose, hemi-cellulose, and lignin to glucose; a usable sugar. We then hypothesized that if the genes coding for these enzymes were expressed in a vector in E. coli, the E. coli would then be able to digest cellulose and create glucose. Finally, we hypothesized that a bioreactor system containing both E. coli and yeast would be able to create ethanol from wood waste as the yeast would ferment the glucose created by the E. coli.</br>
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HOW WE DID IT</br>
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To be assessed later</br>
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<div class="row">
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    <h1 id="FecalSamples" style="color: white;"> Protocols </br></br></h1>
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      <h4><a href="https://static.igem.org/mediawiki/2017/f/fa/Dalfluoro.pdf" style="color: rgba(193,211,93,0.8);"> Fluorophore Assessment of Glycosidase Activity </a></h4>
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      <hr id="hr">
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    <p>Purpose of this protocol is to assess the enzymatic activity of glycosidases using fluorophores conjugated to substrates of interest (such as cellobiose or xylobiose). Once the enzyme of interest cleaves substrate, the fluorophore is released enabling quantification of enzymatic activity.
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<p>
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      <h4><a href="https://static.igem.org/mediawiki/2017/a/af/Dalcosmid.pdf" style="color: rgba(193,211,93,0.8);">Cosmid Library Generation </br></br>
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</a></h4>
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      <hr id="hr">
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    </div>
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    <p>Purpose of this protocol is to generate a library of cosmid clones that are representative of the microbiome of the porcupine for screening for novel enzymes.</br></br></br><p>
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  </div>
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/2/2a/Dalcoomassie.pdf" style="color: rgba(193,211,93,0.8);"> Coomassie Blue Stain </br></br>
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</a></h4>
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      <hr id="hr">
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    </div>
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    <p>To visual total protein in a given sample.</br></br></br></br></br><p>
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  </div>
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<div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/7/79/DalWB.pdf" style="color: rgba(193,211,93,0.8);"> Western Blot
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</a></h4>
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      <hr id="hr">
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    </div>
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    <p>To identify specific amino acid sequences of a protein, or tag, using fluorescently-tagged antibodies.</br></br></br><p>
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  </div>
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/8/89/Dalbatch.pdf" style="color: rgba(193,211,93,0.8);"> Overnight Bacterial Batch Culture
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</a></h4>
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      <hr id="hr">
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    <p>Purpose of this protocol is to grow up single colonies of bacteria for use.</br><p>
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  </div>
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/9/93/Dalcloning.pdf" style="color: rgba(193,211,93,0.8);"> Cloning Overview
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</a></h4>
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      <hr id="hr">
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    <p>Purpose of this overview is to isolate and clone genes of interest into an appropriate plasmid and then introduce the recombinant DNA molecule into <i>E. coli</i>. </br></br>
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<p>
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  </div>
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/7/7a/DalCMC.pdf" style="color: rgba(193,211,93,0.8);"> Congo Red Media Generation </br></br>
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</a></h4>
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      <hr id="hr">
 +
    </div>
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    <p>The purpose of this protocol is to assay carboxymethyl cellulose degradation via a pH change detectable by the dye Congo Red as differentially coloured halos around colonies of bacteria.<p>
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  </div>
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    <div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/1/1b/DalCongored.pdf" style="color: rgba(193,211,93,0.8);"> Congo Red Overlay Assay for Carboxymethylcellulose Degradation
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</a></h4>
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      <hr id="hr">
 +
    </div>
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    <p>Concentration protocol to concentrate DNA isolating using the PowerFecal Extraction kit.</br></br></br><p>
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  </div>
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<div class="col-md-4">
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/9/9d/Dalgelmaking.pdf" style="color: rgba(193,211,93,0.8);"> 0.8% Agarose Gel </br></br>
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</a></h4>
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      <hr id="hr">
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    </div>
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    <p>Purpose of this protocol is to create an agarose gel for visualizing and extracting DNA.</br></br></br><p>
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  </div>
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/e/ee/Dalcelloise.pdf" style="color: rgba(193,211,93,0.8);"> Cellobiose Media Generation
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 +
</a></h4>
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      <hr id="hr">
 +
    </div>
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    <p>The purpose of this protocol is to create solid growth media containing cellobiose to measure beta-glucosidase activity.</br></br></br><p>
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  </div>
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<div class="col-md-4">
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/2/2d/Dalsell.pdf" style="color: rgba(193,211,93,0.8);"> LB Agar Selection Media Plate Generation
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 +
</a></h4>
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      <hr id="hr">
 +
    </div>
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    <p>Purpose of this protocol is to generate solid growth media containing LB and an antibiotic of choice for selective growth of successful clones following transformation.
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<p>
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  </div>
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<div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
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    <div class="row">
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      <hr id="hr">
 +
      <h4><a href="https://static.igem.org/mediawiki/2017/2/24/Daltransform.pdf" style="color: rgba(193,211,93,0.8);"> Heat Shock Transformation
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</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>Purpose of this protocol is to introduce plasmid DNA into <i>E. coli</i> cells.</br></br></br>
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<p>
 +
  </div>
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 +
<div class="col-md-4">
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    <div class="row">
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      <hr id="hr">
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      <h4><a href="https://static.igem.org/mediawiki/2017/7/76/DalCMCCC.pdf" style="color: rgba(193,211,93,0.8);"> Carboxymethylcellulose Media Generation
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</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>The purpose of this protocol is to create solid growth media containing both carboxymethylcellulose and glucose for measuring endoglucanase activity.
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<p>
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  </div>
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<div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
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      <hr id="hr">
 +
      <h4><a href="https://static.igem.org/mediawiki/2017/0/09/Dalsalt.pdf" style="color: rgba(193,211,93,0.8);"> M9 Salts Generation
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</a></h4>
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      <hr id="hr">
 +
    </div>
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    <p>Purpose of this protocol is to generate a salt solution for <i>E. coli</i> growth. </br></br></br><p>
 +
  </div>
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Latest revision as of 23:33, 1 November 2017

Experiments

Protocols

Fluorophore Assessment of Glycosidase Activity

Purpose of this protocol is to assess the enzymatic activity of glycosidases using fluorophores conjugated to substrates of interest (such as cellobiose or xylobiose). Once the enzyme of interest cleaves substrate, the fluorophore is released enabling quantification of enzymatic activity.

Cosmid Library Generation

Purpose of this protocol is to generate a library of cosmid clones that are representative of the microbiome of the porcupine for screening for novel enzymes.


Coomassie Blue Stain

To visual total protein in a given sample.




Western Blot

To identify specific amino acid sequences of a protein, or tag, using fluorescently-tagged antibodies.


Overnight Bacterial Batch Culture

Purpose of this protocol is to grow up single colonies of bacteria for use.

Cloning Overview

Purpose of this overview is to isolate and clone genes of interest into an appropriate plasmid and then introduce the recombinant DNA molecule into E. coli.

Congo Red Media Generation

The purpose of this protocol is to assay carboxymethyl cellulose degradation via a pH change detectable by the dye Congo Red as differentially coloured halos around colonies of bacteria.

Congo Red Overlay Assay for Carboxymethylcellulose Degradation

Concentration protocol to concentrate DNA isolating using the PowerFecal Extraction kit.


0.8% Agarose Gel

Purpose of this protocol is to create an agarose gel for visualizing and extracting DNA.


Cellobiose Media Generation

The purpose of this protocol is to create solid growth media containing cellobiose to measure beta-glucosidase activity.


LB Agar Selection Media Plate Generation

Purpose of this protocol is to generate solid growth media containing LB and an antibiotic of choice for selective growth of successful clones following transformation.

Heat Shock Transformation

Purpose of this protocol is to introduce plasmid DNA into E. coli cells.


Carboxymethylcellulose Media Generation

The purpose of this protocol is to create solid growth media containing both carboxymethylcellulose and glucose for measuring endoglucanase activity.

M9 Salts Generation

Purpose of this protocol is to generate a salt solution for E. coli growth.