Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting

BioTechniques
Balakrishnan RameshNavin Varadarajan

Abstract

Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) typically involves culturing the pooled clones after each sort. In these experiments, if cell viability is compromised by the sort conditions and/or expression of the target protein(s), rescue PCR provides an alternative to culturing but requires re-cloning and can introduce amplification bias. We have optimized a simple protocol using commercially available reagents to directly recover plasmid DNA from sorted cells for subsequent transformation. We tested our protocol with 2 different screening systems in which <10% of sorted cells survive culturing and demonstrate that >60% of the sorted cell population was recovered.

References

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Citations

Sep 22, 2018·Protein Engineering, Design & Selection : PEDS·C S FreiP C Cirino
Nov 12, 2019·ACS Chemical Biology·Balakrishnan RameshNavin Varadarajan
Mar 23, 2021·Biotechnology Progress·Michael VitelliMelih Tamer
Dec 28, 2019·Journal of the American Chemical Society·Tejas NavaratnaGreg M Thurber

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Methods Mentioned

BETA
PCR
FRET
flow cytometry
biosensor

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