FIG 4.

Boot-up of yeast-assembled synthetic S. aureus phage genomes using NEST. (A) The boot-up of either the whole yeast cloned (YC) or yeast assembled (YA) genomes of phage SA75 is depicted. To make SA75YC, cloned SA75 DNA, a PCR-generated repeat fragment, and a linear Ycp/BAC vector harboring terminal homology to the phage DNA and unique restriction site (ISce-I) were transformed into S. cerevisiae (yeast). Similarly, to make SA75YA, TAR-cloned fragments are transformed into yeast with the linear Ycp/BAC vector. DNA from positive clones was transformed into E. coli DH10B cells to produce high concentration plasmid stocks. These plasmids were further transfected/transformed into S. aureus competent cells using the NEST method, incubated at RT for 24 h, and plated using the agar overlay method to observe plaques. (B) To determine the efficiency of boot-up of SA75 DNA isolated from packaged genomes and synthetic genomes, 100 ng of DNA was incubated with 1.13 × 10⁸ CFU of NEST competent cells for 30 min before plating. The efficiency of boot-up was computed by calculating the number of plaques formed per 10⁸ CFU. Each data point represents the mean from three independent experiments, and the error bars indicate standard error.