Figure 5.

Enrichment of S. aureus lytic enzymes by GMD-FACS. Recombinant S. cerevisiae expressing eitheryEGFP::ssLST or LYZ were mixed at a ratio of 1:10,000 and then co-encapsulated with S. aureus. After 16h at 30°C, the colloidal mixture of agarose droplets was stained with SyTox Orange and analyzed by flow cytometry. In both panels, unsorted LYZ GMD are colored blue, unsorted yEGFP::ssLST GMD are green, and GMD falling inside experimental sort gates are orange. A: FACS dot plot of SyTox Orange versus GFP of the presort mixture. Sort gates (vertical bars) were set on the top 1% of SyTox Orange signal. Analysis of GFP signal (region R1) confirms the 10,000:1 input ratio of LYZ yeast to yEGFP::ssLST yeast. Yeast were recovered from sorted GMD, and the process of co-encapsulation, incubation and screening was repeated two more times. B: FACS dot plot of SyTox Orange vs. GFP during the third round sort. By the third round, the GFP positive population has been enriched to 4.57% (region R1). The vast majority of the GMDs falling in the third round SyTox Orange sort gate are yEGFP::ssLST clones (93.7%, as determined by FACS analysis of yeast outgrowth).