A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution

Water-in-oil emulsion
droplets are an attractive format for ultrahigh-throughput
screening in functional metagenomics and directed evolution applications
that allow libraries with more than 107 members to be characterized
in a day. Single library members are compartmentalized in droplets
that are generated in microfluidic devices and tested for the presence
of target biocatalysts. The target proteins can be produced intracellularly,
for example, in bacterial hosts in-droplet cell lysis is therefore
necessary to allow the enzymes to encounter the substrate to initiate
an activity assay. Here, we present a titratable lysis-on-demand (LoD)
system enabling the control of the cell lysis rate in Escherichia
coli. We demonstrate that the rate of cell lysis can be controlled
by adjusting the externally added inducer concentration. This LoD
system is evaluated both at the population level (by optical density
measurements) and at the single-cell level (on single-cell arrays
and in alginate microbeads). Additionally, we validate the LoD system
by droplet screening of a phosphotriesterase expressed from E. coli, with cell lysis triggered by inducer concentrations
in the μM range. The LoD system yields sufficient release of
the intracellularly produced enzymes to bring about a detectable quantity
of product (measured by fluorescence in flow cytometry of double emulsions),
while leaving viable cells for the downstream recovery of the genetic
material.