Mosquitoes are responsible of spreading illnesses such as dengue, malaria and Japanese encephalitis. However, current methods of using chemical pesticides are non-specific in target. Adding further to the problem, over 90 percent of insecticides spread goes to waste as they fail to kill the targets and are subsequently absorbed by the environment instead. Continuation of this kind of practice may trigger ecological and environmental crisis in the future, therefore, a new approach is needed. To rid mosquitoes in an efficient and environmental-friendly way, we have come up with the Mosquito Intelligent Terminator.

The Mosquito Intelligent Terminator is a strain of E. coli that secretes crystal proteins, targetting the wrigglers, larvae of mosquitoes. These crystal proteins from Bacillus thuringiensis are toxic to certain types of mosquitoes only, addressing the problem of specificity. Next, in order to make an environmentally friendly insecticide our design also incorporates a genetic circuit controlling the expression level of cry protein and the population size of E. coli, thus a surplus will never exist as population is self-maintained in this system.

In summary, the Mosquito Intelligent Terminator is designed and optimized to be an ecological and environmental friendly mosquito pesticide.

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I. Cry Protein

Crystal proteins, abbreviated cry proteins, are coded by the cry genes from Bacillus thuringiensis. Each kind of cry protein is highly specific and has different insecticidal toxin to different species of insects.

In our project, we cloned the cry gene from B. thuringiensis subsp. Israelensis HD522. It synthesizes an irregularly shaped parasporal crystal that is highly toxic to certain dipteran larvae, such as Aedes, Culex and Anopheles larvae. Bti parasporal crystalline inclusions contain four major polypeptides with molecular masses of 135, 125, 68 and 28 kDa, referred to as CryIVB, CryIVA, CryIVD, and CytA, respectively, according to the classification designed by Höfte and Whiteley (1989), and belonging to groups Cry4A, Cry4B, Cry11A, and Cyt1A of the new proposed classification (D Dean, personal communication).

Upon ingestion by susceptible insect larvae, the crystal inclusions are solubilized in the alkaline condition of midgut, and the crystal proteins are proteolytically processed by gut proteases into the activated toxin. The toxins activated by gut proteases bind to specific binding sites on the brush border membranes of insect midgut epithelial cells. The conformational change in the toxin molecules triggers the insertion of their pore-forming domain into the membrane. Finally, colloid-osmotic swelling and lysis of the cell result in the death of the larvae.

II. CcdB Protein

From CcdB poisoning of gyrase to death: a model for drug-topoisomerase complex-mediated death. (1) Gyrase binds to DNA (for clarity, wrapping is not represented). (2) When gyrase is cycling on DNA, a "post-strand passage intermediate" is produced. (3) CcdB recognizes and binds this intermediate to form a trapped CcdB-gyrase complex. This CcdB-gyrase-DNA complex is still reversible by the CcdA antidote. (4) The trapped CcdBgyrase complex represents a block for the replication and transcription machineries. Upon collision with a replication fork, this trapped complex becomes irreversible. DNA unwinding by helicases could generate double-strand breaks (6). Step 5 represents an alternative pathway that could occur at high CcdB doses. Stacking of several CcdB molecules could lead to direct dissociation of the gyrase subunits and release of double-strand breaks (as it was proposed for some potent quinolones (Chen et al., 1996)) (6). Step (5) could lead directly to cell death without the need of factors to remove the GyrA subunits that are bound covalently to DNA. However, repair of DNA breaks of step (5) might require unfolding and proteolysis of the GyrA subunits.Apparition of double-strand breaks causes induction of the SOS system. SOS induction mediated by CcdB is recBC dependent (Bailone et al., 1984). Double-strand breaks can be repaired by homologous recombination (7). Abortion of repair will lead to cell death (8).

III. RNA thermometer :BBa_K115002

The expression of heat-shock, cold-shock and some virulence genes is coordinated in response to temperature changes. Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is a widely used regulatory strategy. RNA thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation.

BBa_K115002 is a RNA thermometer that can be used for temperature sensitive post-transcriptional regulation which is based on a RNA thermometer from Salmonella Enterica Tyhpy that initiates translation at 37°C. The switching temperature of the RNA thermometer depends on the stability of the hairpin structure that surrounds the Shine Dalgarno sequence (SD sequence). Melting of the structure at increasing temperature permits ribosome access and translation initiation. At low temperatures, the mRNA adopts a conformation that masks the ribosome binding site (SD sequence) within the 5-untranslated region (5-UTR) and, in this way, prevents ribosome binding and translation.



Fig1. Post-transcriptional regulation acts at the RNA level. At the left, in the off-state, the RNA is folded into a hairpin structure that occludes the Shine Dalgarno region (ribosome binding site). In this situation the translation is blocked because the ribosome cannot bind to the RNA. At the right ,an external factor causes a conformational change of the RNA, exposing the Shine Dalgarno region. Translation can now initiate, because the ribosome is able to bind to Shine Dalgarno region. The system is now in the on-state.




Fig2.The secondary structure of the part, as predicted by RNAfold. At the left, the secondary structure of the original sequences is given. At the right, the secondary structure of the parts, as they are added to the registry, is given. The blue dots around the nucleotides indicate the mutation that was made in order to adjust the secondary structure after introduction of the scar.

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We plan to spread mosquito intelligent terminator in areas where both wrigglers and E. coli thrive, such as areas with still water. By doing so, the E. coli will work as the "terminator" to the mosquito population. This technology is not limited to mosquito's only, with more than one hundred crystal proteins found and characterized, we can create different kinds of E. coli carrying different cry genes to broaden the pesticidal range.

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