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

Abstract

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 10 7 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.

A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

“…Moreover, the assay described here could be modified for implementation in ultrahigh‐throughput format based on droplet microfluidics, with the advantage of detecting real PET hydrolysis products and avoiding the use of surrogate substrates [25] . To that end, the putative workflow comprised of the encapsulation of a PET nanoparticle with the library clone expressing the candidate hydrolase in water‐in‐oil (w/o) droplets prior to total [34,35] or partial [36] cell lysis. After sufficient incubation for PET hydrolysis, the reaction mix proposed in this work was added to the droplets by picoinjection [37,38] or droplet fusion, [39] following a short incubation to reveal the presence of hydrolysis products before proceeding to on‐chip droplet sorting and recovery of the DNA from the droplets by PCR (Figure S9).…”

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

“…146 Adding a level of control, an E. coli system has been introduced that allows for the titratable induction of lysis of a defined fraction of the bacterial population. 240 Alternatively, after bacterial growth, complete lysis can be achieved by picoinjection of lysis agents. 146 Avoiding the need for lysis, enzymes can also be expressed in the bacterial periplasm 82,241 into which many substrates can diffuse, be displayed on the bacterial 122,242 or yeast surface, 124 or be secreted.…”

“…To this end, single cells can be compartmentalized and then grown in droplets . Adding a level of control, an E. coli system has been introduced that allows for the titratable induction of lysis of a defined fraction of the bacterial population . Alternatively, after bacterial growth, complete lysis can be achieved by picoinjection of lysis agents .…”

“…When cells survive the assay, they can be regrown to ultimately produce enough DNA for sequencing. This can be achieved by in-droplet growth followed by partial lysis of cells (leaving enough cells to be recovered), by triggering partial lysis with a kill switch, or by avoiding lysis altogether in display systems (on yeast or E. coli ). Use of high-copy-number plasmids . Near-perfect recovery (80%) can be achieved by employing high-copy-number plasmids in E. coli . ,, Postselection PCR .…”

Smart Droplet Microfluidic System for Single-Cell Selective Lysis and Real-Time Sorting Based on Microinjection and Image Recognition

“…By choice of an appropriate lysis solution, single-cell lysates can be extracted without damage. The first step of microfluidic-based single-cell chemical lysis technology is effective capture, which often utilizes microwalls, , microtraps, microvalves, and independent spaces . The lysis process is then achieved by simply injecting a chemical lysis solution into the channel.…”