|
|
| Line 247: |
Line 247: |
| | <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> | | <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> |
| | </div> | | </div> |
| − | <div class="col-md-4"> | + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <h4><a href="https://static.igem.org/mediawiki/2016/a/a8/T--Dalhousie_Halifax_NS--MetagenomicSequencingAnalysis.pdf"> Metagenomic Sequencing Analysis
| + | |
| − | </a></h4>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="row">
| + | |
| − | <h3 id="FecalSamples" style="color:#4A8A87;">Isolation of Bacteria</h3>
| + | |
| − | </div>
| + | |
| − | <div class="container">
| + | |
| − | <div class="col-md-4">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <h4><a href="https://static.igem.org/mediawiki/2016/1/19/T--Dalhousie_Halifax_NS--CelluloseMediaProtocol.pdf"> Preparation of Cellulose Media </a></h4>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-4">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <h4><a href="https://static.igem.org/mediawiki/2016/c/c5/T--Dalhousie_Halifax_NS--ColonyPCRProtocol.pdf"> 16S Colony PCR </a></h4>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-4">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <h4><a href="https://static.igem.org/mediawiki/2016/6/69/T--Dalhousie_Halifax_NS--ExoSapItProtocol.pdf"> ExoSapIt and DNA Cleanup </a></h4>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="row">
| + | |
| − | <h3 id="FecalSamples" style="color:#4A4D8A;">Metagenomic Library and Transformation</h3>
| + | |
| − | </div>
| + | |
| − | <div class="container">
| + | |
| − | <div class="col-md-6">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <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>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-6">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <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>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <p style="text-align:center;">Protocol for the heat-shock transformation of plasmids into chemically competent <em>E. coli</em> cells.</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="row">
| + | |
| − | <h3 id="FecalSamples" style="color:#4D8A4A;">Chemical Analysis</h3>
| + | |
| − | </div>
| + | |
| − | <div class="container">
| + | |
| − | <div class="col-md-6">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <h4><a href="https://static.igem.org/mediawiki/2016/7/7a/T--Dalhousie_Halifax_NS--steamdistillationprotocol.pdf"> Steam Distillation </a></h4>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-6">
| + | |
| − | <div class="row">
| + | |
| − | <hr id="hr">
| + | |
| − | <h4><a href="https://static.igem.org/mediawiki/2016/f/f1/T--Dalhousie_Halifax_NS--terpeneinhibitionprotocol.pdf"> Terpene Inhibition Experiments </a></h4>
| + | |
| − | <hr id="hr">
| + | |
| − | </div>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
