Bacterial genomes are natural mosaics subject to whole genome engineering by horizontal gene transfer in the wild. Existing approaches to whole-genome engineering have concentrated either on the de novo synthesis of single-source genomes from synthetic DNA, or on small-scale editing at the level of nucleotides to kilobases. The intermediate domain, combining large, megabase-scale segments of DNA from different genomes within the same chromosome for the deliberate construction of chimeric bacterial genomes, has been underexplored. This thesis covers this research gap by presenting methods for constructing megabase-scale bacterial genome chimeras. Chapter 2 presents methods for addition chimera construction using the Additive Conjugative-CAST Engineering (ACE) technology to expand recipient genomes with up to 2-Mb of a donor sequence in a single step. Transfers were demonstrated over large phylogenetic distance, namely 1-Mb cross-order and 100-kb cross-class transfers from Escherichia coli to Vibrio natriegens and to Agrobacterium tumefaciens, respectively. 522-kb cross-order transfers from Pseudomonas protegens to E. coli provide a window to rebooting foreign genome segments in a different genetic background. Chapter 3 extends these methods to substitution chimeras using the technique Replacements by ACE (ReplACE) to rewrite native recipient genome segments with corresponding donor genome segments. We show the recoding of a 1-Mb segment of the E. coli MDS42 genome with E. coli Syn61, and the two-step hybridization of the MDS42 genome with Shigella flexneri to create a Shigoli chimera strain with 50% of its genomic material from both parent strains. We investigate the expression changes from this chimerization event before proceeding to rewrite the genome of MDS42 systematically with that of gut commensal E. coli EcAZ1 to explore genetic determinants of colonization. Chapter 4 articulates the natural barriers of horizontal gene transfer removed with the invention of these two techniques and summarizes the benefits and drawbacks of each method and the biological questions they can address as well as the engineering possibilities they provide. Experimental limitations are considered and future experiments are proposed.
Raymond J. Zhang (Thu,) studied this question.