![]() ![]() C-G: Effects of oil and surfactant combination on the emulsion CFPS. Together with H 2O 2, the active HRP converts the non-fluorescent Amplex Red to fluorescent resorufin. B: The substrate, Amplex Red, and H 2O 2 are delivered into the droplet. A: The HRP is synthesized in the emulsion droplet containing the CFPS mixture. Selection of the appropriate oil/surfactant components during CFPS in emulsion.Ī-B: Scheme of the in situ activity assay for HRP. Bottom: Soluble (S) and insoluble (P) fractions of the HRP were analyzed by SDS-PAGE with subsequent fluorography. The coefficient of variation for each data point was <0.05 (calculated from triplicate measurements). Top: The activity of the HRP in the CFPS mixture measured by the OPD assay. Optimization of the reaction conditions for the CFPS of HRP.Ī: calcium ions concentration. The three-dimensional structure of the horseradish ( Armoracia rusticana) peroxidase isoenzyme C1a (Protein Data Bank: 1ATJ). Step 7: The sequences of the enriched DNA templates are analyzed after several rounds of screening. Step 6: The template DNA on these selected microbeads are amplified to generate a new DNA pool with a higher ratio of the positive clone, and then subjected to another round of screening. The microbeads with the peroxidase mutant of interest and the corresponding DNA are separated according to the relative strengths of their fluorescent intensities. Step 5: After the fluorogenic assay, the microbeads are subjected to a selection process by using FACS (panel F). Then, streptavidin-Cy5 is used to obtain a fluorescent signal from the biotin. During this assay, the peroxidase catalyzes the conversion of the biotin-labeled tyramide to the short-lived tyramide radical that forms a covalent bond with a nearby tyrosine or tryptophan on the HRP surface. Step 4: The enzyme-DNA library on the microbeads is recovered from the emulsion, followed by a tyramide-based fluorogenic assay (panel E POI: protein of interest). In this way, the linkage of the genotype and phenotype on the same microbead is achieved. From the template DNA on the microbead, the peroxidase is synthesized by CFPS in emulsion and immobilized on the same microbead via the scCro-DNA interaction inside the droplets (panel D). Step 3: The recovered microbeads are diluted with the CFPS mixture to less than one microbead per droplet after emulsification. Step 2: The DNA library on the microbeads (panel C) is recovered via emulsion disruption. The template DNA is amplified on the microbead using emulsion PCR (panel B). Step 1: A DNA pool (panel A) is diluted with the PCR mixture containing the primer/scaffold hairpin-immobilized microbeads to less than one template DNA per droplet after emulsification. Scheme showing the principle of the bead display-based uHTS platform for peroxidase. ![]()
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