Figure 2.

Tn7 mutagenesis and high-throughput screening in yeast. (A) Simplified diagram of in vitro Tn7 mutagenesis. The TnsABC^A225V complex enables transposition of mTn7 from its donor plasmid (pMCB82) to its target (pHSS6-based library of yeast genomic DNA), generating a Tn7 insertional library as shown. (B) Screening the Tn7 library in yeast. Ali-quots of the Tn7 insertional library are spread onto plates, and individual bacterial colonies are picked into 96-well plates. Plasmid DNA is prepared from each colony and is individually introduced into a diploid strain of yeast by standard methods of DNA transformation. The resulting yeast transformants are assayed for mTn-encoded β-galactosidase activity using a filter-based assay.