Difference between revisions of "Team:Dalhousie/Experiments"

 
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          <li><a href="https://2017.igem.org/Team:Dalhousie/Description"  >Description</a></li>
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  <h1  style="color:white;"> Experiments </h1>
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<h4 style="text-align:center; color: white;">Background</h4>
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Canada’s forests represent not only an established source of economic revenue, but also a potential source for biofuel substrate. Ligno-cellulosic by-products from pulp and paper processing are removed using chemicals, heat, and water. Microbial cellulose-degrading enzymes are high-value targets for industrial applications. Focused on innovative applications of synthetic biology, the undergraduate Dalhousie iGEM team has undertaken a multi-year project to harness the degradative capacity of microorganisms to convert cellulose into ethanol for biofuel applications. Advances in DNA sequencing technology and bioinformatics have revolutionized our ability to identify useful genes in complex biological samples. We hypothesize that the porcupine microbiome, which includes microorganisms capable of digesting bark and tree resin, will be a rich source of these useful genes. If our hypothesis is correct, mining the porcupine microbiome has the advantage of finding a suite of enzymes that have evolved to work in concert to efficiently degrade cellulose. </p>
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<h4 style="text-align:center; color: white;">The Project</h4>
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Using a combination of metagenomic library construction and bioinformatic analysis we aim to find known cellulolytic enzymes as well as discover novel enzymes.
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Screening of our metagenomic library will allow us to look for a diverse selection of enzymes, including cellulolytic and lignolytic ones by plating on cellulose-only media. The clones that grow can be sequenced for confirmation and cloned into biobricks.
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Our new bioinformatic pipeline now allows us to look for conserved domains rather than known enzymes. This is extremely powerful as it may allow us to discover new enzymes that share catalytic domains with cellulases but are otherwise totally novel.
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The goal of this project is to construct a bioreactor where cellulose would be the input, D-glucose the output and E.coli expressing a suite of cellulolytic enzymes would be the workhorse, converting one to the other. Future goals would be to combine this in co-culture with yeast for the last step in bio-ethanol production.</p>
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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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    <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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      <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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    <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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      <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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    <p>To visual total protein in a given sample.</br></br></br></br></br><p>
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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>
 +
  </div>
 +
 +
    <div class="col-md-4">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <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
 +
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>Purpose of this protocol is to grow up single colonies of bacteria for use.</br><p>
 +
  </div>
 +
 +
<div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <h4><a href="https://static.igem.org/mediawiki/2017/9/93/Dalcloning.pdf" style="color: rgba(193,211,93,0.8);"> Cloning Overview
 +
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <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>
 +
<p>
 +
  </div>
 +
 
 +
<div class="col-md-4">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <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>
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <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>
 +
  </div>
 +
 +
    <div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <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
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>Concentration protocol to concentrate DNA isolating using the PowerFecal Extraction kit.</br></br></br><p>
 +
  </div>
 +
 
      
 
      
−
             
+
 
 +
<div class="col-md-4">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <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>
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>Purpose of this protocol is to create an agarose gel for visualizing and extracting DNA.</br></br></br><p>
 +
  </div>
 +
 
 +
    <div class="col-md-4"style="background-color: rgba(61,85,28, 0.8);">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <h4><a href="https://static.igem.org/mediawiki/2017/e/ee/Dalcelloise.pdf" style="color: rgba(193,211,93,0.8);"> Cellobiose Media Generation
 +
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>The purpose of this protocol is to create solid growth media containing cellobiose to measure beta-glucosidase activity.</br></br></br><p>
 +
  </div>
 +
 
 +
<div class="col-md-4">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <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
 +
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <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.
 +
<p>
 +
  </div>
 +
 
 +
<div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
 +
    <div class="row">
 +
      <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
 +
</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>
 +
<p>
 +
  </div>
 +
 
 +
<div class="col-md-4">
 +
    <div class="row">
 +
      <hr id="hr">
 +
      <h4><a href="https://static.igem.org/mediawiki/2017/7/76/DalCMCCC.pdf" style="color: rgba(193,211,93,0.8);"> Carboxymethylcellulose Media Generation
 +
</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.
 +
<p>
 +
  </div>
 +
 
 +
<div class="col-md-4" style="background-color: rgba(61,85,28, 0.8);">
 +
    <div class="row">
 +
      <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
 +
</a></h4>
 +
      <hr id="hr">
 +
    </div>
 +
    <p>Purpose of this protocol is to generate a salt solution for <i>E. coli</i> growth. </br></br></br><p>
 +
  </div>
 +
 
 +
 
 +
</div>
 +
</div>
 +
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 +
 
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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.