Team:Queens Canada/Project/Attract

We intend to devise a mosquito trap where bacteria will synthesize the chemicals found in foot and body odour. Once the mosquitoes are trapped, we aim to further resolve disease by infecting the mosquitoes to sterilize, control, or destroy the vectors. Inspired by the recent finding of Dr. Fredros Okumu that a synthetic odour blend could be more attractive to a mosquito than a living human, we set out to devise a mosquito trap using synthetic biology. For example, a strain of yeast or bacteria could be engineered to produce CO₂, body odour, and foot odour to lure the insects to their death with relative specificity.

A major roadblock in the eradication of malarial mosquitoes is their tendency to adapt their behavior and develop resistance to the solutions we devise. For example, insecticide-treated mosquito nets are becoming less and less effective because mosquitoes can develop diurnal feeding patterns to avoid them (http://jid.oxfordjournals.org/content/early/2012/09/20/infdis.jis565.abstract). Similarly, the malaria parasite, Plasmodium falciparum can quickly become resistant to new drugs we develop to destroy it. Resistant strains have emerged even for the newest and most potent anti-malarial drug we have at our disposal, artemisinin (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495232/). Our solution circumvents this problem. The same thing that would grant mosquitoes resistance to the traps – i.e. becoming less sensitive to body odour – would also make it more difficult for them to find their human hosts. In this way, it may be possible to slow or even completely prevent mosquitoes from developing resistance to our intervention.

We set out to synthesize indole as our first attractant. We chose the molecule for two reasons. First of all, it is produced endogenously in E. coli so the genes responsible for its synthesis and export, TnaA and TnaB, provided a natural target for PCR amplification. Once amplified, we could then clone the sequences into a plasmid with a strong constitutive promoter to produce the attractant in high yield. The second reason we chose indole is that there are known oderant binding proteins specific for it, and these receptors are highly conserved across mosquito species (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020388/).

In our pathway, TnaB acts as a transporter, allowing for uptake of tryptophan into the bacteria. TnaA then acts as an enzyme to degrade tryptophan to indole. This ultimately allows for the production of indole in cells and if implemented in E. coli could be used to generate large amounts of the compound to act as an attractant.

To that end we started off with using PCR to extract the endogenous genes from E. coli. We were successful in this endeavour, as the gel to the right shows 1.4kb bands which is the appropriate size for TnaA. However unfortunately, our attempts to ligate TnaA, TnaB, and a RBS did not prove successful despite numerous tries. This ended our work with this gene.

If we were to use a species of bacteria in our trap that infected the mosquitoes and specifically affected their biology, such as by inhibiting the synthesis or action of transglutaminase in male mosquitoes, we could potentially do even more to prevent malaria. Inhibition of transglutaminase has been shown to prevent the formation of the male mating plug that is used to keep seminal fluid sealed in place. Because female mosquitoes only mate once in their life, mating with a male infected with the transglutaminase-blocking bacteria would sterilize the female as well, effectively doubling the number of mosquitoes the trap affects.

Infecting mosquitoes with bacteria is not a new concept, and there are a number of options in terms of the chassis that could be used. For example, Lausanne iGEM in 2010 characterized a new chassis, Asaia sp. that can live in the intestinal tract of mosquitoes and be transmitted both horizontally and vertically between mosquitoes. Wolbachia sp. is another strain of bacteria that has been shown to infect the mosquito that carries Dengue fever, Aedes aegipti with good stability (http://www.nature.com/nature/journal/v476/n7361/full/nature10356.html). If this strain could be shown to similarly affect Anopheles gambiae, it might also make an excellent choice of chassis for this purpose.