Synthetic biology approaches for engineering diverse bacterial species

Abstract

When engineers control gene expression, cells can be re-programmed to create living therapeutics or materials by initiating expression of biosynthetic pathways in response to specific signals. In this thesis, two new genetic tools were developed to aid the construction of genetic circuits and facilitate their delivery to bacteria isolated from diverse environments. First, antisense transcription was explored as a new tool for tuning gene expression in Escherichia coli. Antisense transcription was found to reliably repress gene expression and was applied tune simple genetic circuits. Second, an integrative conjugative element from Bacillus subtilis, ICEBsJ, was engineered to deliver exogenous DNA to diverse strains of undomesticated Gram-positive bacteria. Engineered ICEBsI conjugation was demonstrated in twenty different bacterial strains, spanning sixteen species and five genera. To demonstrate ICE's utility in creating new probiotics, the element was used to deliver functional nitrogen fixation pathways (nif clusters) to bacteria isolated from agricultural soils. Collectively, the tools presented here in provide a platform for programing bacteria from diverse environments for advanced applications.