Establishment of a rapid method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome

Abstract

AbstractJCVI-syn3B (syn3B), a minimal synthetic bacterium that only possesses essential genes, facilitates the examination of heterogeneous gene functions in minimal life. Conventionally,Escherichia coliis used to construct DNA fragments for gene transfer into the syn3B genome. However, the construction process is challenging and time-consuming due to various issues, including the inhibition ofE. coligrowth and unexpected recombination, especially with AT-rich DNA sequences such as those found inMycoplasmagenes. Therefore, in this study, we aimed to develop a new transformation method to overcome these issues. We assembled the vector and target DNA fragments using an in vitro homologous recombination system and subsequently transferred the products into the syn3B genome. We obtained approximately 5,000 recombinant colonies per milliliter of the original culture in eight days, which is four days shorter than the conventional period, without any recombination issues, even for AT-rich DNA.SignificanceThe minimal synthetic bacterium JCVI-Syn3B is a useful tool for basic and applied biology. Plasmids for transformation are generally constructed usingEscherichia coli; however, unexpected recombination occurs in some cases. To prevent such issues, this study developed a new method without any in vivoE. colisystem.