High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets

Abstract

The rapid advances of synthetic biology and biotechnology increasingly demand high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase. We present a high-throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limitations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets where fluorescent signals are enhanced by 100 times within 24 h of proliferation. Enrichment of functionally-correct genes by employing error correction method is demonstrated by screening DE droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene. Permeation of isopropyl β-D-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins from at least ~100 bacteria per droplet generates detectable fluorescent signals to enable fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and labor-intensive cell culture typically required in conventional bulk experiment.

Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

“…Microfluidic droplet generation is a powerful technique for encapsulating biological molecules or cells within precisely controlled nL-to pL-volumes, making it possible to perform up to 10 7 reactions in parallel with low per-reaction costs. 1 Microfluidic droplets have been used for a wide variety of applications, including directed evolution of enzymes and proteins, [2][3][4][5][6] digital PCR, 7 large-scale gene assembly, 8 cell culture, 9,10 and, recently, single-cell genomic, epigenomic, and transcriptomic analyses. [11][12][13][14] In the past ten years, droplet technologies have been translated to a variety of commercial assays (e.g.…”

Cross-talk between emulsion drops: how are hydrophilic reagents transported across oil phases?

“…Emulsion drops are often used as vessels to conduct chemical, 1,2 biochemical, 3,4 and biological screening assays at high throughputs. 2,3,[5][6][7][8][9] To achieve a high accuracy, drops must display a narrow size distribution. Drops that fulfil this requirement can be produced with microfluidic devices.…”

An ultrahigh‐throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries

“…This poses a challenge for identifying desired genes using conventional screening methods, such as agar plate assays and microtiter plate assays. Recently, the flow cytometric droplet sorting (FCDS) technique has been recognized as an efficient means for identify novel enzyme variants at the single‐cell level (Chan et al ., 2017; Xu et al ., 2019). FCDS employs monodisperse pico‐litre water‐in‐oil‐in‐water double emulsion droplets as single‐cell micro‐reactors, which encapsulate enzyme‐expressing single cells, cell lysis agent and a fluorogenic substrate.…”

“…Rapidly sorting the droplets based on their fluorescence signals with a commercial flow cytometer (several thousand events per second) allows for isolating enzyme genes with desired properties. Advanced modern flow cytometer instruments significantly bring promise to FCDS‐based screening with their ultrahigh‐throughput multi‐parametric analysis capability, ease of use and wide availability (Lim and Abate, 2013; Zinchenko et al ., 2014; Larsen et al ., 2016; Chan et al ., 2017; Xu et al ., 2019). However, the bottleneck of FCDS exists against generating monodisperse water‐in‐oil‐in‐water double emulsion droplets that are compatible with flow cytometric analysis.…”