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A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-07-14 , DOI: 10.1021/acssynbio.1c00084 Che Fai Alex Wong 1 , Liisa van Vliet 2 , Swapnil Vilas Bhujbal 1 , Chengzhi Guo 2 , Marit Sletmoen 1 , Bjørn Torger Stokke 3 , Florian Hollfelder 2 , Rahmi Lale 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-07-14 , DOI: 10.1021/acssynbio.1c00084 Che Fai Alex Wong 1 , Liisa van Vliet 2 , Swapnil Vilas Bhujbal 1 , Chengzhi Guo 2 , Marit Sletmoen 1 , Bjørn Torger Stokke 3 , Florian Hollfelder 2 , Rahmi Lale 1
Affiliation
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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.
中文翻译：
用于功能宏基因组学和定向进化中液滴分隔超高通量筛选的可滴定细胞按需裂解系统
油包水乳液液滴是功能宏基因组学和定向进化应用中超高通量筛选的一种有吸引力的格式，允许在一天内对超过 10 7个成员的文库进行表征。单个库成员被划分为微流体装置中产生的液滴，并测试目标生物催化剂的存在。靶蛋白可以在细胞内产生，例如，在细菌宿主中，液滴内的细胞裂解是必要的，以允许酶遇到底物以启动活性测定。在这里，我们提出了一种可滴定的按需裂解 (LoD) 系统，能够控制大肠杆菌中的细胞裂解率. 我们证明可以通过调整外部添加的诱导剂浓度来控制细胞裂解的速率。该 LoD 系统在群体水平（通过光密度测量）和单细胞水平（在单细胞阵列和藻酸盐微珠上）进行评估。此外，我们通过对大肠杆菌表达的磷酸三酯酶进行液滴筛选来验证 LoD 系统，细胞裂解由 μM 范围内的诱导剂浓度触发。LoD 系统充分释放细胞内产生的酶以产生可检测量的产物（通过双乳液流式细胞术中的荧光测量），同时留下活细胞用于遗传物质的下游回收。
更新日期：2021-08-20
中文翻译：
用于功能宏基因组学和定向进化中液滴分隔超高通量筛选的可滴定细胞按需裂解系统
油包水乳液液滴是功能宏基因组学和定向进化应用中超高通量筛选的一种有吸引力的格式，允许在一天内对超过 10 7个成员的文库进行表征。单个库成员被划分为微流体装置中产生的液滴，并测试目标生物催化剂的存在。靶蛋白可以在细胞内产生，例如，在细菌宿主中，液滴内的细胞裂解是必要的，以允许酶遇到底物以启动活性测定。在这里，我们提出了一种可滴定的按需裂解 (LoD) 系统，能够控制大肠杆菌中的细胞裂解率. 我们证明可以通过调整外部添加的诱导剂浓度来控制细胞裂解的速率。该 LoD 系统在群体水平（通过光密度测量）和单细胞水平（在单细胞阵列和藻酸盐微珠上）进行评估。此外，我们通过对大肠杆菌表达的磷酸三酯酶进行液滴筛选来验证 LoD 系统，细胞裂解由 μM 范围内的诱导剂浓度触发。LoD 系统充分释放细胞内产生的酶以产生可检测量的产物（通过双乳液流式细胞术中的荧光测量），同时留下活细胞用于遗传物质的下游回收。
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