Project Description

Diseases transmitted by insect vectors, such as malaria and dengue, affect significantly the Brazilian population. In our city, São Carlos, this year in summer the public health organ featured a big number of cases. Working in this problem, our project consists in the development of an alternative repellent of that currently sold in the market and more or equally effective in preventing mosquitoes bites transmitted diseases. The main compound in current repellents is DEET (N, N-diethyl-m-toluamide), a toxic molecule which at certain concentrations could be lethal, and therefore must have strict control on their use in products. The main characteristic of our repellent is the long duration when compared to other products and the replacement of the compound DEET by D-limonene. Instead, the D-limonene has low toxicity, is highly volatile and present a pleasant smell.

Our proposal is building a bacteria carrying out the production of D-limonene via limonene synthase. To enable long term storage at room temperatures, the bacterial cells that make up the repellent will be plasmolyzed, with suspended metabolism, considered a dormant state. The maintenance of this state will be obtained by a solution of polyethyleneglycol (PEG) that will raise the osmotic pressures. Once in contact with the skin, the PEG solution will be diluted by sweat, inducing an osmotic shock in cells. Then the universal stress protein promoter (UspA) will be activated and will induce the expression of limonene synthase. Beyond PEG, other compounds such as glycerol and metal ions in low concentrations will compose our insect repellent cream, in order to sustain the bacteria to the required metabolites building and posterior enzyme activities. In this way, the distribution of our repellent may become feasible, allowing its use.

To overcome those problems with limonene synthase folding, we used constitutive promoters for the expression of chaperones from all Escherichia coli available classes (like ClpB, DnaK and IbpA / IbpB). It will reinforce the stockpile of chaperones naturally produced during osmotic shocks (heat-shock proteins) in bacteria. Our goal is to improve protein solubility, indirectly creatinga toolkit for protein solubility enhancement for future iGEM teams. . Besides, reducing the occurrence of insoluble bodies and improving the production of limonene.

Finally, a way to ensure the biosafety in the use of our repellent and keep a control of bacterial cells is the use of a system of programmed death or Kill Switch. This system will work with two suicide genes: Killer red, a protein which trigger oxidative species generation destroying the DNA (plasmid and chromosomal) allied to a potent RNAse which will avoid any contribution of RNA to host or normal microbiota. These genes will be switch on through an association with a zinc sensitive promoter triggered by zur proteins which associate with zinc and avoid the expression of them allowing the bacterial growing. After long periods of repellent action, bacteria activities will decrease zinc concentration in medium, which allows the gene activation and cells death.

Plasmolisis

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Limonene Synthase

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Manufacturing

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