Team:StanfordBrownSpelman/Material Waterproofing

Material Waterproofing

Primary approach: Paper wasp protein

While cellulose-based biomaterials have promising applications in aeronautics and various other fields due to their lightweight, biodegradable nature, they risk structural failure if they absorb too much water from wet environments. Fortunately, nature has provided us with a potential solution. Paper wasps of the genus Polistes are well known for their ability to collect cellulose from the plants around them, mix it with their saliva, and use the resulting cement to construct nests with paper-like properties.

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. We believe there may be a single protein in wasp saliva that is responsible for the hydrophobic nature of their nests. We collected Polistes dominula, an invasive European species of paper wasp, and sequenced the proteins found in their nests using peptide mass fingerprinting. Our ultimate goal is to transform the gene coding for the wasp waterproofing protein into Saccharomyces cerevisiae so that we can produce an inherently biomimetic solution to shielding 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.

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.

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.

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!

Material Waterproofing Project Notebook: Complete documentation of our methods, protocols, and scientific processes can be found in the linked document.

Results

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.

References

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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