Team:StanfordBrownSpelman/Material Waterproofing


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Stanford–Brown–Spelman iGEM 2014 — Amberless Hell Cell

Polistes dominula, also known as the European paper wasp. The nest paper is a durable mixture of saliva and cellulose pulp.
The most significant property of the wasp-produced paper is that it is hydrophobic and therefore waterproof. Research has shown that the nest paper is composed primarily of cellulose, but coated with a protein-rich oral secretion CITATION NEEDED. Until now, scientific knowledge of this protein coating was limited mainly to total amino acid composition of all the proteins in the paper. To gain more insight into the specific proteins that may exist in wasp nest paper, we collected Polistes dominula, an invasive European species of paper wasp, and sequenced the proteins found in their nests using peptide mass fingerprinting. We believe there may be a single protein in wasp saliva that is chiefly responsible for the hydrophobic nature of their nests. Our ultimate goal is to identify the gene that codes for this wasp waterproofing protein and transform into Saccharomyces cerevisiae so that we can produce an inherently biomimetic solution to shield lightweight bacterial cellulose (BC) or bacterial cellulose acetate (BCOAc) films from water in the environment. This project is particularly exciting because of its potential for discovery; never before have the proteins in wasp saliva been identified or applied as functional biomaterials.
Alternate approach: Wax ester biosynthesis
The biodegradable unmanned aerial vehicle (UAV) would be best improved if it had waterproofing capabilities. As such, various waterproofing mechanisms are under investigation for application [1]. One of the mechanisms includes the biological manipulation of the protein involved in the secretion of lipophilic wax esters from the avian uropygial gland of a pelican. Previous research has revealed that the chemical composition of the uropygial gland secretion is primarily composed of unique variations of methylhexanoic acid and fatty alcohols that react to produce wax esters. The enzymes responsible for catalyzing the esterification reaction are wax synthases. Various wax synthases have been identified across many eukaryotic and prokaryotic organisms including plants, mammals, protozoa, and bacteria. However, the current focus is bacterial and protozoan production of wax esters. Bacterial production of wax esters is most commonly associated with the Acinetobacter calcoaceticus bacterium and isoprenoid wax ester production in Marinobacter hydrocarbonoclasticus [2-3]. M. hydrocarbonoclasticus and Euglena gracilis, a bacterium and protist respectively, were the primary focus for the synthesis of wax esters. There were two proteins that efficiently catalyzed the production of isoprenoid wax esters: wax synthase 1 and wax synthase 2. Both of these proteins can be found in M. hydrocarbonoclasticus. Therefore, we used molecular biology to investigate the biosynthetic production of wax esters in E. coli for waterproofing capabilities.
  • 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 a wasp 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.
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