Generating double emulsions on two chips and selection of active
biocatalysts. The workflow for one cycle of directed evolution consists
of the following steps: (i) Gene libraries are generated from an enzyme-encoding
plasmid. (ii) E. coli cells produce
the biocatalyst of interest in liquid culture. (iii) In a first microfluidic
device (with hydrophobic, fluorocarbon-coated channel walls), single
cells are compartmentalized in droplets together with substrate and
lysis agents. (iv) After cell lysis, substrate and cytoplasmically
expressed enzyme react to yield a fluorescent product. (v) The reaction
is allowed to proceed for a desired incubation period (in our case
up to 24 h, but droplets are stable for at least one month). The reaction
progress can be stopped simultaneously in all water-in-oil droplets
by heat inactivation, so that the time required for double emulsion
formation and sorting does not extend the assay period. (vi) Next,
primary droplets are transformed into double emulsions in a second
device with identical design to the one used in (iii) but with hydrophilic
coating. (vii) Variants exhibiting the highest activity are identified
and sorted in a standard flow cytometer. The recovered DNA can be
used for further rounds of evolution without PCR amplification when
a high-copy plasmid is used. The procedure takes little time: droplet
formation (steps iii and vi) takes place at a frequency of 6–12
kHz, so that a library of 107 double emulsion droplets
is produced in 90 min. Sorting 107 droplets at a rate of
10–15 kHz takes about 15 min.