In this talk, I will discuss our investigations into two key areas: (i) Recapitulating and testing endosymbiotic theory-based organelle evolution in laboratory setting, and (ii) engineering and evolving synthetic auxotrophs as live bacterial vaccine candidates. The origin of organelles is one of the key outstanding questions in the evolution of eukaryotic organisms. Based on the endosymbiotic theory, eukaryotic organelles like mitochondria and chloroplasts are proposed to have originated and evolved from bacterial endosymbionts during an early stage of evolution; sequencing studies spanning several decades have supported this hypothesis. However, there is minimal understanding (if any) of how bacterial endosymbionts evolved and transformed into organelles. In this talk, I will discuss our synthetic approaches to experimentally model mitochondria and chloroplast evolution in laboratory setting. We modeled the first stage of mitochondrial evolution by engineering endosymbiosis between two genetically tractable model organisms, E. coli and S. cerevisiae. In this model system, we engineered E. coli strains to survive in the yeast cytosol, and provide ATP to a respiration-deficient yeast mutant. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Similarly, I will also describe our initial efforts involving engineering cyanobacterial endosymbionts in yeast cells as a step towards recapitulating chloroplast evolution. These readily manipulated systems should allow us to investigate various aspects of the endosymbiotic theory of organelle evolution. In the next part of this talk, I will describe our directed evolution strategies to generate synthetic auxotrophs as live bacterial vaccine candidates. Our approach will be to build synthetic, orthogonal biochemical pathways essential for growth in derivatives of virulent bacteria and then test these rationally designed and evolved "synthetic" organisms as live bacterial vaccine candidates.
