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Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting
Balakrishnan Ramesh1, Christopher S Frei1, Patrick C Cirino1
1Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX.
Biotechniques
|September 9, 2015
Summary
Researchers developed a new method to recover plasmid DNA directly from sorted cells, bypassing culturing. This technique improves recovery rates for screening expressed protein libraries, especially when cell viability is low.
Area of Science:
- Molecular Biology
- Biotechnology
- Protein Engineering
Background:
- Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) commonly requires culturing sorted clones.
- Low cell viability post-FACS, due to sort conditions or protein expression, can hinder traditional methods.
- Existing alternatives like rescue PCR can introduce bias and require re-cloning.
Purpose of the Study:
- To optimize a protocol for direct plasmid DNA recovery from FACS-sorted cells.
- To provide an alternative to cell culturing that minimizes bias and re-cloning steps.
- To enhance the efficiency of screening expressed protein libraries.
Main Methods:
- Developed a simple protocol using commercially available reagents for direct plasmid DNA recovery.
- Applied the protocol to FACS-sorted cells from two different screening systems.
- Quantified plasmid DNA recovery and assessed its suitability for subsequent transformation.
Main Results:
- Successfully optimized a protocol for direct plasmid DNA recovery from sorted cells.
- Demonstrated >60% recovery of the sorted cell population.
- The method proved effective even when <10% of sorted cells survived culturing.
Conclusions:
- The developed protocol offers a robust alternative to culturing for plasmid DNA recovery after FACS.
- This method significantly improves the recovery of genetic material from compromised cell populations.
- It streamlines the screening of expressed protein libraries, particularly in challenging experimental conditions.
