title: Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening.
abstract: In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins.
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, China.
authors: Fuqiang
authors: F
authors: Ma
authors: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
authors: Michael
authors: M
authors: Fischer
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, China.
authors: Yunbin
authors: Y
authors: Han
authors: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
authors: Stephen G
authors: SG
authors: Withers
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, China.
authors: Yan
authors: Y
authors: Feng
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai 200240, China.|Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), East China University of Science and Technology , 130 Meilong Road, Shanghai 200237, China.
authors: Guang-Yu
authors: GY
authors: Yang
mesh_terms: D003374:Coumarins / Q000737:chemistry; D005434:Flow Cytometry; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D057927:Hydrophobic and Hydrophilic Interactions; D015394:Molecular Structure; D010316:Particle Size; D015202:Protein Engineering; D011506:Proteins / Q000032:analysis*
chemical_list: D003374:Coumarins; D011506:Proteins; C030123:coumarin