Team:StanfordBrownSpelman/Material Waterproofing


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Stanford–Brown–Spelman iGEM 2014 — Amberless Hell Cell

  • Our first, successful gel containing proteins from the three paper wasp nest we collected. The presence of several strong bands indicated that the waterproofing effect was likely the result of the interactions of only a few key proteins.

  • Frozen wasp paper sample collected during the summer from a live but vacated nest.

  • A paper wasp on the nest we cultivated on the roof of our lab at the NASA Ames Research Center. Our personal wasp nest provided us with the freshest possible samples.

  • Protein samples extracted from three paper wasp nests collected with Dave Kavanaugh, entomologist from the California Academy of Sciences.

  • Jotthe Kannappan grinds a frozen wasp in the process of extracting RNA.

  • A macroscopic photo of one of our paper wasps used for species identification.

  • Paper wasp actively working on building its nest.
Approach & Methods
Our approach to identifying the Polistes dominula waterproofing protein relied on the acquisition of nest samples and of individual wasps. Our plan, detailed in the graphic below, was to extract protein from the nest samples and use analytical techniques such as peptide mass fingerprinting to gather information on the proteins present, and then to use this information to identify candidate Polistes dominula genes for cloning and testing in model organisms.

Figure 1. Schematic for wasp nest protein identification via peptide mass fingerprinting.
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 dominula 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.

A short time lapse video documenting the running of one of our our wasp nest protein gel..

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.

Wasp candidate genes.
1. Biester, EM et al. (2012) Identification of avian wax synthases. BMC Biochemistry 13:4. PMID: 22305293.

2. Kalscheuer, R & Steinbüchel, A (2003) A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J. Biol. Chem. 278(10):8075-82. PMID: 12502715.

3. Holtzapple, E & Schmidt-Dannert, C (2007) Biosynthesis of Isoprenoid Wax Ester in Marinobacter hydrocarbonoclasticus DSM 8798: Identification and Characterization of Isoprenoid Coenzyme A Synthetase and Wax Ester Synthases. J. Bacteriology 189:3804-3812. PMID: 17351040.
Additional Information
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