Publications

Robust and highly efficient transformation method for a minimal mycoplasma cell

Mizutani M, Glass JI, Fukatsu T, Suzuki Y, Kakizawa S

PMID: 39903184

Abstract

Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here, we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining the genome of . When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, colony forming unit, and transformation efficiency, it was found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early exponential growth phase. Notably, the transformation efficiency of up to 4.4 × 10 transformants per cell per microgram of plasmid DNA was obtained. A method to obtain several hundred to several thousand transformants with less than 0.2 mL of culture with approximately 1 × 10-10 cells and 10 ng of plasmid DNA was developed. Moreover, a transformation method using a frozen stock of transformation-ready cells was established. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells.