Team:StanfordBrownSpelman/Material Waterproofing

From 2014.igem.org

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<h6>We then narrowed our list down to six proteins with favorable PSI-BLAST hits and obtained the DNA sequences for these genes from the genome. Three of these genes were codon-optimized and synthesized for E. coli expression in the pF1A T7 Flexi® vector, while the other three were amplified from wasp RNA via RT-PCR for S. cerevisiae expression in the pYES2.1/V5-His-TOPO® vector.</h6>
<h6>We then narrowed our list down to six proteins with favorable PSI-BLAST hits and obtained the DNA sequences for these genes from the genome. Three of these genes were codon-optimized and synthesized for E. coli expression in the pF1A T7 Flexi® vector, while the other three were amplified from wasp RNA via RT-PCR for S. cerevisiae expression in the pYES2.1/V5-His-TOPO® vector.</h6>
  
  
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<div class="sub4"><a href="http://docs.google.com/document/d/1jpWJ48e9fPUjGYJ3KOXdE5UEZzHpWGWydRSrWBVN_Kg/edit?usp=sharing" target="_blank"><h6><img src="http://2014.igem.org/wiki/images/2/25/SBS_iGEM_2014_download.png"></a><a href="http://docs.google.com/document/d/1jpWJ48e9fPUjGYJ3KOXdE5UEZzHpWGWydRSrWBVN_Kg/edit?usp=sharing"><b>Material Waterproofing Project Notebook:</b> Complete documentation of our methods, protocols, and scientific processes can be found in the linked document.</a></h6></div>
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<div class="sub4"><a href="http://2014.igem.org/wiki/images/3/36/WaspProteins.pdf" target="_blank"><h6><img src="http://2014.igem.org/wiki/images/2/25/SBS_iGEM_2014_download.png"></a><a href="http://docs.google.com/document/d/1jpWJ48e9fPUjGYJ3KOXdE5UEZzHpWGWydRSrWBVN_Kg/edit?usp=sharing"><b>Material Waterproofing Project Notebook:</b> Complete documentation of our methods, protocols, and scientific processes can be found in the linked document.</a></h6></div>
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Revision as of 00:02, 18 October 2014

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.

Photo Reel
  • A polyacrylamide gel containing proteins from the paper wasp nests we collected. We excised the dominant bands for protein analysis.

  • Frozen wasp paper sample collected during the summer from an active 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]. We ran the proteins on two polyacrylamide gels, one with a ten minute 70ºC heat denaturation step and the other without. We then 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.


Our initial plan was to extract RNA from female nest-building wasps so we could purify their messenger RNA, generate a complementary DNA library, and get the library sequenced for use as a reference transcriptome. 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. The genome was used as a reference for peptide mass fingerprinting, we saved our RNA extracts for eventual RT-PCR amplification, and the project moved onwards. We truly live in an exciting time for genetic engineering!
Once we obtained the mass fingerprinting data, we were faced with a long list of uncharacterized peptide fragments with hits in the Polistes dominula genome. We used the NCBI Position-Specific Iterated Basic Local Alignment Search Tool, or PSI-BLAST, to search online databases for proteins with similar sequences and domains. This gave us hints towards the possible functions of these uncharacterized proteins.
We then narrowed our list down to six proteins with favorable PSI-BLAST hits and obtained the DNA sequences for these genes from the genome. Three of these genes were codon-optimized and synthesized for E. coli expression in the pF1A T7 Flexi® vector, while the other three were amplified from wasp RNA via RT-PCR for S. cerevisiae expression in the pYES2.1/V5-His-TOPO® vector.
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.


Wasp candidate genes.

Alternate approach: Wax ester biosynthesis
In addition to the wasp protein waterproofing project, we searched literature for other organisms that would be able to produce a highly hydrophobic substance to waterproof the UAV. We stumbled across Marinobacter hydrocarbonoclasticus, a marine bacterium that synthesizes isoprenoid wax esters for use as storage compounds CITATION NEEDED.


A phytol-based wax ester produced by Marinobacter hydrocarbonoclasticus.
Wax esters, such as the example shown above, consist of two very long carbon chains joined by an ester group. These chains impart great hydrophobicity to the entire molecule, indicating that a wax ester coating on a surface could be used to repel water. We decided to repurpose the wax ester synthesis genes in Marinobacter to create a pathway through which Escherichia coli could synthesize the wax esters and use them to waterproof our UAV.
The two enzymes in Marinobacter that are involved in the production of isoprenoid wax esters were wax synthase 1 (WS1) and wax synthase 2 (WS2). In the presence of a fatty alcohol and fatty acyl-CoA (carboxylic acid derivative), either of these two proteins can catalyze the synthesis of isoprenoid wax esters.
The DNA sequences for both WS1 and WS2 were obtained from the NCBI database CITATION NEEDED. They were then codon optimized for Escherichia coli using a codon optimizer from the Integrated DNA Technologies (IDT) website and then synthesized. After the genes were received from IDT, a polymerase chain reaction (PCR) was done to amplify the proteins and to ensure that they were present at around 1.4kb. The proteins were then ligated to a promoter and RBS and also into a chloramphenicol vector, then transformed into E. coli. After completing a colony PCR on the transformants, colonies 5, 7, and 11 on the WS2 plate appeared to contain the WS2 gene. Liquid cultures were inoculated with colonies 5, 7, and 11 to create more cells with the gene. After the cultures were grown in the incubator, they were miniprepped in order to extract their plasmid DNA. The DNA was then sent for sequencing with a 15ul reaction volume.
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:


WS2 sequencing alignment in Geneious 7.
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 could ever make its 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.
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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