low-binding 96-well microtiter plates  (Greiner Bio)

Journal: ACS Catalysis

Article Title: Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor

doi: 10.1021/acscatal.4c00230

Figure Lengend Snippet: Overview of the screening approach. (a) TrpB-expressing cells are encapsulated in single emulsion droplets with the aptamer sensor, the substrates (Ser, indole), and lysis reagents. (b) One —Lysis reagents will release TrpB in each droplet where a cell is present. Two —TrpB catalysis of Ser and indole to produce Trp. Three —The fluorescent aptamer is initially self-quenched, but once Trp is bound in favor of the quenching complementary strand, the sensor lights up and becomes fluorescent. The Trp concentration measured as a fluorescence signal is a function of the catalytic efficiency, stability, and expression strength of TrpB variants, and screening and selection can be carried out accordingly. (c) Droplets are encapsulated again into double emulsion droplets so that they are compatible with fluorescence-activated cell sorting on a commercial flow cytometer. (d) Genotype from the pool of highly fluorescent droplets is recovered, after which the enriched pool of active variants is rescreened in the microtiter plate-based format for single variants of interest. The chip design is shown in Supplementary Figure 1 .

Article Snippet: The aptamer sensor was combined 1:1 either with purified chemicals or with TrpB reaction mixtures in a final volume of 80 μL in low-binding microtiter plates (Corning, 3881).

Techniques: Expressing, Emulsion, Lysis, Concentration Assay, Fluorescence, Selection, Double Emulsion, FACS, Flow Cytometry

Journal: ACS Catalysis

Article Title: Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor

doi: 10.1021/acscatal.4c00230

Figure Lengend Snippet: Overview of the screening approach. (a) TrpB-expressing cells are encapsulated in single emulsion droplets with the aptamer sensor, the substrates (Ser, indole), and lysis reagents. (b) One —Lysis reagents will release TrpB in each droplet where a cell is present. Two —TrpB catalysis of Ser and indole to produce Trp. Three —The fluorescent aptamer is initially self-quenched, but once Trp is bound in favor of the quenching complementary strand, the sensor lights up and becomes fluorescent. The Trp concentration measured as a fluorescence signal is a function of the catalytic efficiency, stability, and expression strength of TrpB variants, and screening and selection can be carried out accordingly. (c) Droplets are encapsulated again into double emulsion droplets so that they are compatible with fluorescence-activated cell sorting on a commercial flow cytometer. (d) Genotype from the pool of highly fluorescent droplets is recovered, after which the enriched pool of active variants is rescreened in the microtiter plate-based format for single variants of interest. The chip design is shown in Supplementary Figure 1 .

Article Snippet: On the next day, the reaction mixture was allowed to cool down, and 30 μL was mixed with 30 μL 2× aptamer sensor stock solution in low-binding microtiter plates (Corning, 2881).

Techniques: Expressing, Emulsion, Lysis, Concentration Assay, Fluorescence, Selection, Double Emulsion, FACS, Flow Cytometry