Title: Development of genetically intact bioengineered spores of Bacillus subtilis
Description: Genetic engineering tools are under continuous development. However, hesitation by consumers and governments regarding consumption of genetically modified organism (GMO) affects taking advantage of developments in biotechnology. While being a complicated issue to address, this challenge inspired us to investigate whether it is possible to engineer organisms without altering their wild-type genomes, but with the same customizability level offered by genetic engineering; that is, having the capacity of expressing foreign proteins not codified by the wild-type genome. I used B. subtilis spores as a model organism for this purpose. I took advantage of the sporulation process during which two compartments with differential expression, or different gene expression patterns co-exist, the mother cell and the forespore, and I programmed a single designer plasmid to behave differently in each compartment: the plasmid in the mother cell modifies the spore phenotype, while the plasmid in the forespore undergoes self-digestion.

At the end of sporulation, the mother cell lyses and releases the final product — a plasmid-free engineered spore. Following this, I incorporated the forespore-specific "self-digestion" gene circuit into a variety of plasmids with different purposes, including the generation of spores expressing GFP on their protective coats and the artificial induction of sporulation, both of them as a proof-of-concept of genetically intact bioengineered organisms. Production of the different types of genetically intact bioengineered spores resulted in an average of nearly 90% of them free of detectible plasmid or genome alterations. Spores of B. subtilis and other species overall continue to gain attention in the biotechnology sector, with potential applications ranging from biopesticides, probiotics, and vaccines to energy-converting materials, self-healing concrete, and whole-cell biocatalysts. While spores represent a special case of multiple-compartment organisms among bacteria, most eukaryotic organisms possess multiple compartments, structures, or tissues with differential expression, including plants and animals.

Therefore, our results in this study could serve as a starting point for new ideas and methods for the genetic modification-free engineering of complex organisms or parts of them.