"
Team:StanfordBrownSpelman/Material Waterproofing
From 2014.igem.org
(Difference between revisions)
| Line 197: | Line 197: | ||
<h6>After sequencing the data came back with miniprepped genes that aligned with the original synthesized gene for roughly half the length of the sequence, but with a puzzling truncation that was present in all three plasmid samples:</h6> | <h6>After sequencing the data came back with miniprepped genes that aligned with the original synthesized gene for roughly half the length of the sequence, but with a puzzling truncation that was present in all three plasmid samples:</h6> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<h6>Unfortunately, the wax esters project had to be discontinued due to an error in the synthesis of the WS2 gene. In order to insert the gene into the vector, there are restriction sites that are cleaved by their respective restriction enzymes. When we codon-optimized WS2 for E. coli expression, we accidentally created a PstI cut site at base 720 in the synthesized gene. Because there was a cut site in the middle of the gene, only a fragment of the WS2 gene ever made their way into the chloramphenicol vector, thereby disabling the protein the gene would have coded for. We also discovered that the fatty acyl-CoA reagent we intended to use for our enzymatic synthesis reaction was exorbitantly expensive (over $60 per milligram). For this reason, and because it would have been too costly to resynthesize the WS2 gene, the project was discontinued.</h6> | <h6>Unfortunately, the wax esters project had to be discontinued due to an error in the synthesis of the WS2 gene. In order to insert the gene into the vector, there are restriction sites that are cleaved by their respective restriction enzymes. When we codon-optimized WS2 for E. coli expression, we accidentally created a PstI cut site at base 720 in the synthesized gene. Because there was a cut site in the middle of the gene, only a fragment of the WS2 gene ever made their way into the chloramphenicol vector, thereby disabling the protein the gene would have coded for. We also discovered that the fatty acyl-CoA reagent we intended to use for our enzymatic synthesis reaction was exorbitantly expensive (over $60 per milligram). For this reason, and because it would have been too costly to resynthesize the WS2 gene, the project was discontinued.</h6> | ||
Revision as of 16:20, 17 October 2014
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. This poses a problem for anyone who wishes to fly our UAV on a rainy day. 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.
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. This poses a problem for anyone who wishes to fly our UAV on a rainy day. 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.
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.
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!
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.
