Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting.
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.
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX.
Balakrishnan
B
Ramesh
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX.
Christopher S
CS
Frei
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX.
Patrick C
PC
Cirino
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX.
Navin
N
Varadarajan
D015374:Biosensing Techniques; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D019151:Peptide Library; D010455:Peptides / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011753:Pyrones / Q000032:analysis
C518065:ARAC peptide; D019151:Peptide Library; D010455:Peptides; D011753:Pyrones; D049452:Green Fluorescent Proteins; C030720:triacetic acid lactone; D004247:DNA; D002918:Chymotrypsin; C117609:chymotrypsin B
library screening; plasmid recovery; toxicity