Difference between revisions of "Team:Dalhousie/Experiments"

 
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     <h3 id="FecalSamples" style="color:#674A8A;"> High-Throughput Sequencing </h3>
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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/2016/d/d6/T--Dalhousie_Halifax_NS--PowerFecalProtocol.pdf"> Fecal Sample DNA Extraction </a></h4>
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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>To extract environmental DNA from fecal samples. This extraction will purify genomic DNA from prokaryotic and eukaryotic cells found in the fecal sample.  
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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/2016/7/70/T--Dalhousie_Halifax_NS--PowerFecalConcentration.pdf"> DNA Concentration</a></h4>
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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>Concentration protocol to concentrate DNA isolating using the PowerFecal Extraction kit.<p>
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     <p>To visual total protein in a given sample.</br></br></br></br></br><p>
 
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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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    <p>To identify specific amino acid sequences of a protein, or tag, using fluorescently-tagged antibodies.</br></br></br><p>
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       <h4><a href="https://static.igem.org/mediawiki/2016/a/ae/T--Dalhousie_Halifax_NS--16IlluminaSeq.pdf"> 16S Illumina Miseq Sequencing </a></h4>
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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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     <p>Illumina MiSeq Sequencing was used to identify the sequences of the 16S rRNA genes in the fecal samples. This step was undertaking by the Integrated Microbiome Resource at Dal. There protocol was provided to us.<p>
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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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       <h4><a href="https://static.igem.org/mediawiki/2016/4/46/T--Dalhousie_Halifax_NS--MetagenomicSequencingIllumina.pdf"> Metagenomic Shotgun Illumina Miseq Sequencing </a></h4>
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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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     <p>This protocol was used by the Integrated Microbiome Resource to sequence the metagenomic DNA in our fecal samples from the porcupine, beaver, arctic wolf and coyote.
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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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       <h4><a href="https://static.igem.org/mediawiki/2016/d/d1/T--Dalhousie_Halifax_NS--16SSequencingAnalysis.pdf"> 16S Sequencing Analysis
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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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     <p>This protocol was used for analyzing the 16S Illumina Sequencing data from the illumina sequencer. Provided by the Integrated Microbiome Resource at Dalhousie University.<p>
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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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       <h4><a href="https://static.igem.org/mediawiki/2016/a/a8/T--Dalhousie_Halifax_NS--MetagenomicSequencingAnalysis.pdf"> Metagenomic Sequencing Analysis
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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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     <p>This protocol was used for analyzing the Metagenomic Illumina Sequencing data from the illumina sequencer. Provided by the Integrated Microbiome Resource at Dalhousie University.<p>
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     <p>Concentration protocol to concentrate DNA isolating using the PowerFecal Extraction kit.</br></br></br><p>
 
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<h3 id="FecalSamples" style="color:#4A8A87;">Isolation of Bacteria</h3>
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  <h4><a href="https://static.igem.org/mediawiki/2016/1/19/T--Dalhousie_Halifax_NS--CelluloseMediaProtocol.pdf"> Preparation of Cellulose Media </a></h4>
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  <p>Congo Red Cellulose media can be used to isolate bacteria that degrade cellulose. It does this because the only carbon source in the media is cellulose. It uses Whatman Paper and gelatin.</p>
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       <h4><a href="https://static.igem.org/mediawiki/2016/c/c5/T--Dalhousie_Halifax_NS--ColonyPCRProtocol.pdf"> 16S Colony PCR </a></h4>
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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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     <p>Colony PCR has many applications including; verifying that an insert has been successfully incorporated in plasmid constructs, insert orientation, and species determination of unknown samples. The later was the reason for our use of Colony PCR. After streaking fecal sample onto cellulose plates, we picked colonies of different morphologies to find out which species or genus they were. We did this by using primers that targeted conserved sequence found in bacteria so we could amplify the DNA of almost all bacteria. </p>
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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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       <h4><a href="https://static.igem.org/mediawiki/2016/6/69/T--Dalhousie_Halifax_NS--ExoSapItProtocol.pdf"> ExoSapIt and DNA Cleanup </a></h4>
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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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     <p>Instead of PCR clean up, we used ExoSap It. ExoSap It allows for an enzymatic clean-up of left over dNTPs and single stranded DNA. It is a very quick procedure as the only activation ExoSap It needs is a 15 minute 37°C incubation and a 15 minute 80 °C inactivation.</p>
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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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  <h3 id="FecalSamples" style="color:#4A4D8A;">Metagenomic Library and Transformation</h3>
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  <h4 style="text-align:center;"><a href="https://static.igem.org/mediawiki/2016/c/c3/T--Dalhousie_Halifax_NS--MetagenomicLibraryProtocol.pdf" style="text-align:center;"> Metagenomic Library Construction </a></h4>
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  <p style="text-align:center;">Protocol that takes extracted fecal DNA, prepares it for ligation and clones it into a cosmid. This cosmid can be packaged into phage particles and those can be used to place the cosmid into <em>E. coli</em> cells which will express environmental DNA that had been placed into the cosmid.</p>
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  <h4 style="text-align:center;"><a href="https://static.igem.org/mediawiki/2016/0/02/T--Dalhousie_Halifax_NS--Transformation.pdf">Heat-Shock Transformation</a></h4>
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  <p style="text-align:center;">Protocol for the heat-shock transformation of plasmids into chemically competent <em>E. coli</em> cells.</p>
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  <h3 id="FecalSamples" style="color:#4D8A4A;">Chemical Analysis</h3>
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  <h4><a href="https://static.igem.org/mediawiki/2016/7/7a/T--Dalhousie_Halifax_NS--steamdistillationprotocol.pdf"> Steam Distillation </a></h4>
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  <p>Steam Distillation was used to separate the terpenes from pine tree oleoresin without degrading them. Through GC/MS analysis we confirmed that this procedure worked.</p>
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       <h4><a href="https://static.igem.org/mediawiki/2016/f/f1/T--Dalhousie_Halifax_NS--terpeneinhibitionprotocol.pdf"> Terpene Inhibition Experiments </a></h4>
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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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     <p>These experiments were used to test <em>E. coli</em> and <em>S. cerevisiae</em> tolerance to terpenes, both in pure form and the terpenes isolated from oleoresin.</p>
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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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      <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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    <p>Purpose of this protocol is to introduce plasmid DNA into <i>E. coli</i> cells.</br></br></br>
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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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    <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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      <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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    <p>Purpose of this protocol is to generate a salt solution for <i>E. coli</i> growth. </br></br></br><p>
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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.