Paratransgenesis is a technique in which symbiotic bacteria are engineered to secrete anti-pathogenic molecules within vector species, thereby altering the phenotype of the host to control zoonotic diseases. Asaia bogorensis, an alpha-proteobacteria naturally found in the midgut of Anopheles mosquitoes, can be modified to secrete anti-plasmodial effectors. This technique has the capability to reduce the incidence of malaria, the most prolific insect-vectored disease in the world. Previous work has developed anti-plasmodial strains of A. bogorensis. However, the anti-plasmodial effector genes are present on plasmids with antibiotic resistance markers, which make them unstable in field conditions. To ensure plasmid stability in the field, a method for host-plasmid dependency must be implemented into A. bogorensis without the use of antibiotic resistance. To this end, we utilized a method known as auxotrophic gene complementation. This is accomplished by first deleting a gene in the chromosome that encodes a biochemical precursory necessary for survival of A. bogorensis using homologous recombination. We intend to delete the essential gene, dapA, from A. bogorensis. The gene, dapA, is an auxotrophic gene compatible with diaminopimelic acid-enriched media. Without dapA, the bacterium will die. In the future, we can supplement the dapA survival gene in trans via plasmid, which will allow the auxotrophic A. bogorensis to survive. This translocation of dapA from the genome to plasmid is essential for a stable host-plasmid dependency method in the field.
