The Project

Diseases transmitted by insect vectors, such as malaria, dengue fever, yellow fever and leishmaniasis, affect millions of people and caused about 600.000 deaths around the world in 2012, especially in tropical regions(1). In Brazil, population is affected significantly by those diseases. In the summer of 2014/2015 there were 1.350.406 cases of dengue in Brazil, resulting in 614 deaths (57% higher than the same period in 2013/2014) (2). Brazilian health authorities affirmed that the dengue fever epidemic had a raise of 229% (3). In our city, Sao Carlos, the epidemic was so severe that the public health agency has not been able to account for the total number of cases (link vigilância epidemiológica). Unofficial reports count more than 20.000 victims and at least 5 dead people (4, 5).

Dengue fever is an urban disease, caused by 4 different types of viruses. Those viruses are transmitted by female mosquitoes Aedes aegypti bites (6,7). Most of times, symptoms can be confused with a bad cold. In severe cases, however, patients exhibit haemorrhagic fever, which can lead to death. There is not specific treatment or vaccine to prevent dengue fever (8). The only actions that may help avoiding the disease are controlling the environment where the insects reproduce and using repellents. Every year, we see the same sad situation repeat itself, so far without prospects for change.

Considering this problem, our project consisted in developing an alternative repellent of that currently sold in the market, more or equally effective in preventing mosquitoes bites that transmits 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 its use (9). The main characteristic of our repellent is a longer duration when compared to other products and the replacement of DEET by D-limonene, a less toxic compound.

Our proposal was 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 would be plasmolyzed, with suspended metabolism, in a dormant state. The maintenance of this state would be obtained by a solution of polyethylene glycol (PEG) that will raise the osmotic pressure. Once in contact with the skin, PEG solution would be diluted by sweat and inducing osmotic shock in bacteria cells. Then the universal stress protein promoter (UspA) would be activated, inducing expression of limonene synthase. In addition to PEG, other compounds such as glycerol and metal ions in low concentrations would constitute our insect repellent cream, in order to keep bacteria at the required metabolic condition and posterior enzyme activities. This way, the distribution of our repellent may become feasible, allowing its use.

The expression of limonene synthase was the aim of other iGEM team (TU_Munich, 2012). However, protein folding has been an unsolved problem. To overcome those hindered limonene synthase folding, we used constitutive promoters for expression of chaperones from all Escherichia coli available classes (like ClpB, DnaK and IbpA / IbpB). It would reinforce chaperones stocks naturally produced during osmotic shocks (heat-shock proteins) in bacteria. Besides improving limonene production, our goals were to improve protein solubility, indirectly creating a toolkit for protein solubility enhancement for future iGEM teams, reducing the occurrence of insoluble bodies.

Finally, we proposed a system of programmed death - a killswitch - to ensure biosafety and biosecurity of the engineered bacteria. The mechanism evolves two suicide genes: Killer Red and Barnase. The first consists in a protein that triggers the generation of reative oxygen species, destroying both plasmid and chromosomal DNA. The second results in a protein with ribonuclease activity. The genes would be activated by a zinc sensitive promoter triggered by zur proteins that, when associated with zinc, inhibit killswitch expression, allowing bacteria to grow. After some hours of repellent action, bacteria activities would decrease zinc concentration in medium, leading to gene activation and, thus, to death of the bacteria.