Robust and highly efficient transformation method for a minimal Mycoplasma cell

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 of the genome of Mycoplasma mycoides. When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, CFU, and transformation efficiency, we 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, we attained the transformation efficiency up to 4.4 x 10-2 transformants per cell per g plasmid DNA. We developed a method to obtain several hundred to several thousand transformants with less than 0.2 mL of cultured cells and 10 ng of plasmid DNA. Moreover, we established a transformation method using frozen stock of transformation-ready cells. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCEMycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand cellular process of life and for bioengineering because of their simple metabolism, small genomes and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtainable only 10 nanograms of plasmid DNA, which is around one-thousandth amount required for traditional mycoplasma transformations. Moreover, we established a convenient method using frozen stocks of transformation-ready cells. These improved methods play a crucial role in further studies using minimal cells.