Team:StanfordBrownSpelman/Material Waterproofing

From 2014.igem.org

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<h6>We extracted total protein from the nest samples using a plant protein extraction kit [CITATION NEEDED]. After denaturing the proteins and running them on a polyacrylamide gel, we excised all dominant individual bands and sent them to Dr. Gary Wessel’s lab at Brown University for peptide mass fingerprinting.</h6>
<h6>We extracted total protein from the nest samples using a plant protein extraction kit [CITATION NEEDED]. After denaturing the proteins and running them on a polyacrylamide gel, we excised all dominant individual bands and sent them to Dr. Gary Wessel’s lab at Brown University for peptide mass fingerprinting.</h6>
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<div class="small-7 small-centered columns"><br><center><img src="https://static.igem.org/mediawiki/2014/9/9e/SBSiGEM2014_Wasp_Nest_Sample.jpg"><br>
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<h6><center>A small sample of a <i>Polistes eominula</i> nest waiting to be ground with a mortar and pestle for protein extraction.</center></h6>
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<h6>Peptide mass fingerprinting is an analytical protein identification technique in which the protein of interest is cleaved into small fragments via site-specific proteolytic enzymes such as trypsin. The molecular masses of these fragments can be measured accurately through mass spectrometry. Once these masses are known, they can be compared with computer predictions based on a reference genome or transcriptome to see which of the reference’s proteins are most abundant.</h6>
<h6>Peptide mass fingerprinting is an analytical protein identification technique in which the protein of interest is cleaved into small fragments via site-specific proteolytic enzymes such as trypsin. The molecular masses of these fragments can be measured accurately through mass spectrometry. Once these masses are known, they can be compared with computer predictions based on a reference genome or transcriptome to see which of the reference’s proteins are most abundant.</h6>

Revision as of 23:08, 16 October 2014

Stanford–Brown–Spelman iGEM 2014 — Amberless Hell Cell

Approach & Methods
Our approach to identifying the Polistes dominula waterproofing protein relied on the acquisition both of nest samples and of individual wasps. Our plan, detailed in the graphic below, was to use analytical techniques to gather information on proteins in the nest samples, and then to use this information to identify candidate genes for cloning and testing in model organisms.
We extracted total protein from the nest samples using a plant protein extraction kit [CITATION NEEDED]. After denaturing the proteins and running them on a polyacrylamide gel, we excised all dominant individual bands and sent them to Dr. Gary Wessel’s lab at Brown University for peptide mass fingerprinting.


A small sample of a Polistes eominula nest waiting to be ground with a mortar and pestle for protein extraction.
Peptide mass fingerprinting is an analytical protein identification technique in which the protein of interest is cleaved into small fragments via site-specific proteolytic enzymes such as trypsin. The molecular masses of these fragments can be measured accurately through mass spectrometry. Once these masses are known, they can be compared with computer predictions based on a reference genome or transcriptome to see which of the reference’s proteins are most abundant.
Fortunately for us, the Polistes dominula genome was published shortly after we began our project [CITATION NEEDED], saving us the trouble and extreme expense of sequencing wasp RNA to create a reference transcriptome ourselves. We truly live in an exciting time for genetic engineering!
From the peptide mass fingerprinting data, we obtained a list of thirty fragments with hits in the wasp genome. After running a PSI-BLAST on the amino acid sequences of each fragment to look for similar, characterized sequences in related species, we chose six genes as candidates for the waterproofing protein.


Figure 1. Figure caption here.
Results
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References
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Additional Information
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