Fig. 3. Droplet flow cytometry for molecular evolution. (A) Schematic workflow of directed enzyme evolution by screening and selecting positive DE droplets via FACS. Left: (1) a variant gene library was transformed and cloned into E. coli, (2) the encoded proteins translated within E. coli, (3) encapsulation of single cells in the W/O emulsion droplets, (4) addition of fluorogenic substrates through the oil phase and the formation of W/O/W DE droplets. Cells with functional enzymes can convert the non-fluorescence substrates into fluorescent products entrapped in the internal core, (5) analysis of enzymatic activity across the DE droplet populations via FACS. Right: FACS results for DE droplets containing E. coli expressing wide-type (wt) enzyme, unselected library (R0) and the library after one (R1), two (R2) and three (R3) rounds of sorting based on gate M1. Adapted with permission from ref. 54. Copyright 2006 Cell Press. (B) Schematic workflow of directed enzyme evolution using gel-shell beads (GSBs) and FACS. (1) Encapsulation of single E. coli cells expressing target enzyme in single emulsions and cell lysis to liberate the enzyme and its coding plasmid, (2) release of a fluorescent product by catalysis, (3) formation of GSB to entrap products, (4) high-throughput screening and sorting of GSB containing catalytically active hits via FACS, (5) recovery of variants with desired phenotypes, (6) iterative rounds of selection. Adapted with permission from ref. 93. Copyright 2014 Nature Publishing Group.