Team:Queens Canada/Project/Repel

Our idea of a mosquito repellent aims to neutralize the odours that mosquitoes are attracted to. Recent studies have shown that mosquitoes infected with the malarial parasite, Plasmodium falciparum, are four times more attracted to foot odour than non-infected mosquitoes. By removing the volatile compounds that cause these smells, we hypothesize that this will effectively decrease the ability of mosquitoes to find humans. Our project thus focuses on creating a genetic and enzymatic pathway to remove those volatile compounds. It begins with the uptake of isovaleric acid, a known mosquito semiochemical that is present is foot odour, and converts it into banana smell. Domestically, this serves as a foot deodorant but on a global scale it may also curb malaria infection rates.

AtoE is a non-specific membrane transporter of short-chain fatty acids endogenously expressed in ''E.coli''. High constitutive expression of this gene would allow high intake of surrounding isovaleric acid and channelling into the breakdown pathway.

In order to isolate the gene from the E. coli genome, we designed PCR primers so that the AtoE gene could be replicated with the correct biobrick prefix and suffix. The size of the AtoE gene is 1323 bp, but with the addition of the standard biobrick cut sites (43bp), a 1.4kb band was expected and was seen on the gel of the PCR products (on right).

Carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (NPT) are endogenously expressed in Nocardia iowensis. NCAR is a highly non-specific carboxylic acid reductase that converts fatty acids such as isovaleric acid into aldehydes. NPT is an enzyme that induces a post-translational modification by adding a pantetheinyl arm to the active site of the carboxylic acid reductase, which is crucial for the formation of a thioester intermediate. NCAR was submitted as a single part by Calgary iGEM 2012, but it is dependent on NPT for full function. We thus sought to link these two enzymes together to improve Calgary's part.

YjgB is a non-specific zinc-containing alcohol dehydrogenase endogenously expressed in E. coli. It reduces a number of different aldehydes to their corresponding alcohol using NADPH as a co-substrate. In this instance, we planned to use yjgB to reduce isoamyl aldehyde to isoamyl alcohol. We were successful in amplifying yjgB by PCR, cloning it into pSB1C3 with a high constitutive expression cassette, producing high concentrations of protein at the predicted size of 36kDa on an SDS PAGE gel. We verified our biobrick before sending it into the registry and found the sequence to be an exact match for the putative sequence from GenBank. Over the next week we will be using mass spectrometry and an NADPH activity assay to verify the amino acid sequence of the part and quantify its specific activity towards isobutyraldehyde. This is the part we have submitted this year. More details on this can be found on the Parts page on the wiki.

Alcohol acetyl-transferase 1 (ATF1) (BBa_J45014) is an enzyme endogenous to Saccharomyces cerevisiae and was first used by MIT iGEM 2006. It catalyzes the conversion of isoamyl alcohol to isoamyl acetate, which has a banana smell. This will complete the neutralization of isovaleric acid. We received the ATF1 gene from the Biobrick registry where we transformed and cut the part to be placed in with other genes in our pathway.

Caenorhabditis elegans has impressive chemotaxis behaviour which is naturally used to seek out food sources. It has been shown to respond to a wide range of both water-soluble and volatile chemicals as either attractants or repellants with a GPCR system.

In order to test the effectiveness of our part, it was necessary to create an assay to test our completed part against. Using the assay methodology pioneered by QGEM 2011 (Margie et. al. 2013), worms were placed in a common starting area and their movement in response to a control substance and to the test odorant was observed. With an anaesthetic at target sites to immobilize worms at the end of each trial, the number of worms in the test and control quadrants were counted to calculate a chemotactic index. More details on how this assay works can be outlined here.

Isovaleric acid solutions at 1M, 7:1, and 99% concentrations were used. A total of 38 chemotaxis assay plates were run. The graph to the right shows our results from the standard graph for our chemotaxis assay. According to statistical analysis, there was no significant difference between C. elegans motility response to the control and test samples.