Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation,Lab on a Chip

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Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation
Lab on a Chip ( IF 6.1 ) Pub Date : 2020-11-12 , DOI: 10.1039/d0lc00830c
Paul Jannis Zurek ₁ , Raphaëlle Hours , Ursula Schell , Ahir Pushpanath , Florian Hollfelder

Affiliation

Microfluidic ultrahigh-throughput screening of enzyme activities provides information on libraries with millions of variants in a day. Each individual library member is represented by a recombinant single cell, compartmentalised in an emulsion droplet, in which an activity assay is carried out. Key to the success of this approach is the precision and sensitivity of the assay. Assay quality is most profoundly challenged when initially weak, promiscuous activities are to be enhanced in early rounds of directed evolution or when entirely novel catalysts are to be identified from metagenomic sources. Implementation of measures to widen the dynamic range of clonal assays would increase the chances of finding and generating new biocatalysts. Here, we demonstrate that the assay sensitivity and DNA recovery can be improved by orders of magnitude by growth of initially singly compartmentalised cells in microdroplets. Homogeneous cell growth is achieved by continuous oxygenation and recombinant protein expression is regulated by diffusion of an inducer from the oil phase. Reaction conditions are adjusted by directed droplet coalescence to enable full control of buffer composition and kinetic incubation time, creating level playing field conditions for library selections. The clonal amplification multiplies the product readout because more enzyme is produced per compartment. At the same time, phenotypic variation is reduced by measuring monoclonal populations rather than single cells and recovery efficiency is increased. Consequently, this workflow increases the efficiency of lysate-based microfluidic enzyme assays and will make it easier for protein engineers to identify or evolve new enzymes for applications in synthetic and chemical biology.

中文翻译：

超高通量微滴筛选中的生长扩增可提高克隆酶测定的灵敏度，并最大程度地减少表型变异

酶活性的微流超高通量筛选每天可提供具有数百万个变体的文库信息。每个单独的文库成员均由在乳胶小滴中隔开的重组单细胞代表，在其中进行活性测定。该方法成功的关键是测定的准确性和灵敏性。当要在定向进化的早期轮次中增强最初的薄弱，混杂活动或要从宏基因组学来源中鉴定出全新的催化剂时，测定质量将受到最大的挑战。采取措施扩大克隆测定的动态范围将增加发现和产生新的生物催化剂的机会。这里，我们证明了通过微滴中最初单个分隔的细胞的生长，可以提高检测灵敏度和DNA回收率几个数量级。均质细胞的生长是通过连续的氧合作用实现的，重组蛋白的表达是通过诱导剂从油相的扩散来调节的。通过直接的液滴聚结来调节反应条件，以完全控制缓冲液的组成和动力学温育时间，从而为文库选择创造了公平的竞争环境。克隆扩增使产物读出倍增，因为每个隔室产生更多的酶。同时，通过测量单克隆种群而不是单个细胞减少了表型变异，并且提高了回收效率。所以，

更新日期：2020-12-10

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