DEVELOPING MOLECULAR TOOLS FOR GENOME-WIDE EDITION OF VIBRIO GENOMES

GOMES DE OLIVEIRA, ANDREY GIOVANNI ; LETICIA LAROTONDA; LLORENTE, BRIARDO; ALFONSO SOLER BISTUE

Congreso; SAMIGE 2022; 2022

SAMIGE

The genus Vibrio includes more than 70 bacteria species. Some, such as V. cholerae, are well-known pathogens. Others, like V. natriegens, have great biotechnological potential due to their fast growth and other useful traits such as natural competence and efficient secretion of heterologous proteins. Although methods to engineer Vibrio bacteria have been developed, there is still a lack of molecular tools to enable genome-wide modifications. Here we present two approaches we are developing for genome-wide scale engineering in Vibrio bacteria. The first method is a loxP/Cre system that enables genome insertions, replacements, and deletions of genetic parts. We transformed V. natriegens with synthetic DNA constructs bearing antibiotic resistance genes flanked by loxP sites and homologousregions targeting the nuclease gene dns to generate kanamycin, chloramphenicol, zeocin, andspectinomycin resistant strains. Then, we used genomic DNA from these strains to transform and attempt targeted-genome edits in strains containing different antibiotic resistance genes. These experiments resulted in cells that swapped their antibiotic resistance genes at the targeted dns locus. To demonstrate the robustness and versatility of the approach, we successively swapped the antibiotic resistance genes for four additional rounds and also removed them from the genome. The second method uses recombineering tools to relocate genes to targeted genome loci. Using V. cholerae, we relocated the ATP synthase locus encoding the motor protein responsible for the production of ATP. We hypothesized that the relocation of this gene from its conserved position close to the oriC of chromosome I to other parts of the genome would affect the physiology of the bacterium. Via natural transformation and homologous recombination, we flanked the ATP synthase locus with the AttL and AttR sites of the HK bacteriophage and introduced an attB site close to the ter region. Then, we successfully relocated the ATP locus from the oriC to the ter region via transient expression of Int and Xis recombinases. The presented approaches now enable targeted genome-wide insertions, replacements, deletions, and relocations of genetic parts in Vibrio bacteria.