Efficient Natural Plasmid Transformation ofVibrio natriegensEnables Zero-capital Molecular Biology

Abstract

The fast-growing microbe Vibrio natriegens is capable of natural transformation where it draws DNA in from media via an active process under physiological conditions. Using an engineered strain with a genomic copy of the master competence regulator tfoX from Vibrio cholera in combination with a new minimal competence media (MCM) that uses acetate as an energy source, we demonstrate naturally competent cells which are created, transformed, and recovered entirely in the same media, without exchange or addition of new media. Cells are naturally competent to plasmids, recombination with linear DNA, and co-transformation of both to select for scarless and markerless genomic edits. The entire process is simple and inexpensive, requiring no capital equipment for an entirely room temperature process (Zero Capital protocol, 10^4 cfu/μg), or just an incubator (High Efficiency protocol, 10^5-6 cfu/μg). These cells retain their naturally competent state when frozen and are transformable immediately upon thawing like a typical chemical or electrochemical competent cell. Since the optimized transformation protocol requires only 50 minutes of hands-on time, and V. natriegens grows quickly even on plates, a transformation started at 9 AM yields abundant culturable single colonies by 5 PM. Further, because all stages of transformation occur in the same media, and the process can be arbitrarily scaled in volume, this natural competence strain and media could be ideal for automated directed evolution applications. As a result, naturally competent V. natriegens could compete with E. coli as an excellent chassis for low-cost and highly scalable synthetic biology.

Simplified post-assembly plasmid library amplification for increased transformation yields inE. coliandS. cerevisiae

“…To further extend the utility of our approach, we confirmed its use for large library creation in S. cerevisiae , a highly useful organism in biotechnology and synthetic biology for which RCA-mediated library amplification has not been explored previously. We expect that the product of our protocol could be of benefit in organisms other than E. coli or S. cerevisiae , such as the rapidly developing biotechnology chassis organism V. natriegens 28 for which efficient and scalable natural transformation techniques have been established 29 . Additionally, the linear DNA product of our protocol could be of use for genome integration rather than plasmid formation.…”

Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity

“…To maximize our chances of evolving a high-performing biosorption microbe, we decided to work with the extremely fast-growing and highly engineerable microbe Vibrio natriegens . − As increased mutation rates tend to be associated with an accumulation of growth defects, we hypothesized that starting with a faster growing organism could make these defects relatively less severe. Recent work has also succeeded in making V. natriegens naturally competent, a feature that is very useful for conducting targeted mutagenesis once the ideal genetic targets in V. natriegens for REE biosorption are better understood …”

“…Recent work has also succeeded in making V. natriegens naturally competent, a feature that is very useful for conducting targeted mutagenesis once the ideal genetic targets in V. natriegens for REE biosorption are better understood. 31 …”