An expanded genetic toolbox to accelerate the creation ofAcholeplasma laidlawiidriven by synthetic genomes

ABSTRACT Assembling synthetic bacterial genomes in yeast and genome transplantation has enabled an unmatched level of bacterial strain engineering, giving rise to cells with minimal and chemically synthetic genomes. However, this technology is currently limited to members of the Spiroplasma phylogenetic group, mostly Mycoplasmas , within the Mollicute class. Here, we propose new genetic tools for developing these technologies for Acholeplasma laidlawii , which is phylogenetically distant from Mycoplasmas and, unlike most Mollicutes , uses a standard genetic code. We first investigated a donor-recipient relationship between two A. laidlawii strains through whole-genome sequencing. We then created multi-host shuttle plasmids and used them to optimize an electroporation protocol. We also demonstrated the use of evolution to create superior strains for DNA uptake via electroporation. For genome transplantation, we selected A. laidlawii 8195 as the recipient strain and created a PG-8A donor strain by inserting a Tn5 transposon carrying a tetracycline resistance gene. The tools presented here will improve Acholeplasma research and accelerate the effort toward creating A. laidlawii strains driven by synthetic genomes. Graphical Abstract