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. 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.

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

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]. 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.

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.

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

We collected two wasp nests in the field: one new nest that we saw wasps actively working on, and an older, abandoned nest that Dr. Kavanaugh estimated to be about one year old. After extracting total protein from the nests, we ran the protein samples on two polyacrylamide gels – one with a ten minute 70ºC heat denaturation step and the other without – and obtained the images below.

The presence of a few dominant bands in the protein samples indicated that there may be a single protein chiefly responsible for the hydrophobicity of wasp nest paper. The relative faintness of bands from the older nest also suggests that the protein may degrade over time, which supports our qualitative observations that the older nest was somewhat less waterproof.

Once we obtained peptide mass fingerprinting data on the bands we excised from the protein gels, we were faced with a list of thirty uncharacterized proteins whose fragments had hits in the Polistes dominula genome. Interestingly, the proteins present in each gel slice did not always have the sizes we expected in comparison to the protein ladder. Why this was the case remains to be seen, but we postulate that proteins of different affinities traveled through the gel at different speeds, may have complexed and traveled more slowly, or have been so abundant that traces were present in multiple bands.

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.

Nearly all of the PSI-BLAST results came back with strong functional predictions for each of our proteins, the majority of which we deemed irrelevant to waterproofing ability. For example, many proteins appeared to be variants of ubiquitin, a small regulatory protein found in virtually all eukaryotic tissues. Others were similar to vitellogenin, an egg yolk precursor protein used to nurture larvae in related insect species such as Vespula vulgaris (also known as the common yellow-jacket) and Formica exsecta (narrow-headed ant). While it was interesting to see the diversity of proteins present in the wasp nests, we had to narrow our scope to test candidate genes in the lab.

CLICK HERE to download a .ZIP file containing the results of our peptide mass fingerprinting, as well as a document listing the thirty proteins present, their first PSI-BLAST hits, predicted sizes, and amino acid sequences.

Out of this list of thirty proteins present in the protein gel slices, we narrowed our list down to six candidate proteins with favorable PSI-BLAST hits. The genes that coded for these candidate proteins are what we ultimately ended up working on in the lab. These six proteins are detailed in the table below.

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.

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:

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 et al. (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 et al. (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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