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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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| + | <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
Material Waterproofing
Primary approach: Paper wasp protein
Paper wasps of the genus Polistes are well known for their ability to construct nests out of transformed plant materials with paper-like properties. The most significant property of the wasp-produced paper is that it is hydrophobic and therefore waterproof. Research has identified that a protein found in the salivary glands of paper wasps is responsible for coating, strengthening, and thus waterproofing the cellulose found in plants. We collected Polistes dominula, an invasive European species of paper wasp, and sequence the proteins found in their saliva using a modified peptide mass fingerprinting approach. 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, bacteria and protozoa 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.
Primary approach: Paper wasp protein
Paper wasps of the genus Polistes are well known for their ability to construct nests out of transformed plant materials with paper-like properties. The most significant property of the wasp-produced paper is that it is hydrophobic and therefore waterproof. Research has identified that a protein found in the salivary glands of paper wasps is responsible for coating, strengthening, and thus waterproofing the cellulose found in plants. We collected Polistes dominula, an invasive European species of paper wasp, and sequence the proteins found in their saliva using a modified peptide mass fingerprinting approach. 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, bacteria and protozoa 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 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.
