Advances and applications of droplet-based microfluidics in evolution and screening of engineered microbial strains

doi:10.12211/2096-8280.2021-105

Abstract

Abstract:

Engineered microbial strains are the important foundation of biofabrication. However, most strains usually need to be engineered to improve their performances to be used in industry. Therefore how to efficiently screen and isolate the improved target strains is a critical step of strain engineering. As an advanced high-throughput screening technology using micro-chip, droplet-based microfluidics technology can generate highly independent and uniformed micro- or nano- liter droplets, in which single cells can be encapsulated, inoculated, detected, and isolated for strain engineering. It is especially useful in the evolution of strains for producing extracellular products. In this review, we first introduced the basic components of the droplet-based microfluidic system and the main steps involved in strain screening. We then summarized the application of the droplet-based microfluidic technology in strain engineering according to the key factors of the technology such as the signal sources of droplet detection, the difficulties of handling droplet screening, and the application scopes of droplet sorting instruments. According to the instruments required for the droplet sorting, we concluded the application cases into two types either via fluorescence-activated droplet sorting (FADS) using microfluidic equipment or via fluorescence-activated cell sorting (FACS) using flow cytometry instrument. The FADS type using single-layer water-in-oil droplet can be further classified into cellular signature, fluorescent reporter protein, and substrate-based reaction according to the signal sources, while the FACS type can be divided into double-layers water-in-oil-in-water (W/O/W) droplet or microgel droplet according to the droplet property. At last, we outlined the challenges and prospects of the droplet microfluidic technology and provided some guidelines for its applications in synthetic biology. Compared with traditional screening methods such as shaking flask or microplate with a throughput of hundreds to thousands of samples per day in milli- or micro- liter volume, the droplet-based microfluidic technology can achieve millions of samples per day in pico- or nano- liter volume, resulting in thousand-folds increased screening speed and million-folds decreased consumption cost. By integrating with an automated station, the droplet-based microfluidic technology can be further promoted for its screening efficiencies and application potentials in microbial synthetic biology.

Key words: droplet-based microfluidics, single-cell analysis, cell factory, high-throughput screening, direct evolution

CLC Number:

Q939.97

Ran TU, Shixin LI, Haoni LI, Meng WANG. Advances and applications of droplet-based microfluidics in evolution and screening of engineered microbial strains[J]. Synthetic Biology Journal, doi: 10.12211/2096-8280.2021-105.

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液滴类型	液滴信号来源	优缺点	通量	代表性应用案例
单层液滴	细胞自身信号（荧光、拉曼、浊度、细胞图像）	优点：操作简单，细胞直接包埋在液滴中。缺点：需要特殊软件，例如拉曼和图像识别软件。	荧光300 Hz；拉曼1~5 Hz；图像识别5~10 Hz；浊度240 Hz	色素：核黄素^[103]；拉曼：虾青素^[78]；图形识别：人成红细胞白血病细胞^[106]；散射光：细胞浊度^[82]
	细胞荧光蛋白（目标蛋白和外源荧光蛋白融合、生产菌自身含生物传感器、生产菌和感应菌共包埋培养）	优点：操作简单，细胞直接包埋在液滴中。缺点：需要构建特定生物传感器。	150~300 Hz	生产菌自身含生物传感器：3-脱氢莽草酸^[107；生产菌和感应菌共培养：香豆酸酵母菌和大肠杆菌感应报告菌^[109]
	细胞和底物（同时共包埋在液滴）	优点：操作简单，细胞和底物直接包埋在液滴中。缺点：不适合酶促反应快的样品。	300~2000 Hz	大肠杆菌：半乳糖苷酶^[92]、硫酯酶^{[47, 56]}等；枯草芽孢杆菌：淀粉酶^[114]；丝状真菌：纤维素酶^[119]
	细胞和底物（细胞先包埋在液滴，底物后加入）	优点：可以通过液滴融合添加试剂，用于酶促反应快的样品。缺点：液滴融合操作复杂。	300~1000 Hz	漆酶^[112]、脂肪酶^[122]、乙醇^[123]等

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