Development of a Novel Shuttle Vector for Tetragenococcus halophilus

Abstract

Tetragenococcus halophilus is used by companies in a variety of fermented foods while also being shown to be capable of being a potent probiotic. T. halophilus fermentative batches are at risk of being compromised by viral pathogens, which leads to product loss in the industry. To address the need for a molecular toolset that was capable of being maintained in T. halophilus that expresses desired genes, a plasmid (pCBW2) was designed and built in E. coli. With T. halophilus having no prior optimized way of transformation, multiple methods of transformation were tested in an attempt to introduce pCBW2 to T. halophilus. The plasmid pCBW2 was built using PCR and restriction enzymes to insert a combination of preexisting reporter elements paired with hypothetical promoters from T. halophilus genomic DNA. After construction, chemical transformation, electroporation, and biolistic transformation methods were attempted under a variety of conditions to introduce the constructed plasmid to T. halophilus. While no attempts of the transformation of T. halophilus were successful, conventional chemical transformation of E. coli was successful. E. coli were able to utilize promoters endogenous to T. halophilus, suggesting pCBW2 is a functional plasmid capable of expressing provided genes of interest.