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Flow cytometric screening of cDNA expression libraries for fluorescent proteins
Paul H Bessette1, Patrick S Daugherty
1Department of Chemical Engineering, and Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, California 93106, USA.
Biotechnology Progress
|June 5, 2004
Summary
Fluorescence-activated cell sorting (FACS) enabled the discovery of novel, brighter green fluorescent proteins from rare bacterial cDNA libraries. This method efficiently identifies and purifies fluorescent proteins with unique properties.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Screening cDNA expression libraries for rare genes is challenging.
- Fluorescent proteins are valuable tools in molecular biology.
- Existing fluorescent proteins have limitations in brightness and properties.
Purpose of the Study:
- To apply Fluorescence-activated cell sorting (FACS) for high-throughput screening of bacterial cDNA libraries.
- To identify and characterize novel fluorescent proteins from rare sources.
- To evaluate the performance of newly isolated fluorescent proteins compared to existing ones.
Main Methods:
- Construction of a cDNA expression library from Astrangia lajollaensis in bacteria.
- Quantitative screening using Fluorescence-activated cell sorting (FACS) to detect rare fluorescent cells.
- Purification and characterization of isolated fluorescent proteins, including brightness and chromophore analysis.
Main Results:
- Rare fluorescent cells (1 in 200,000) were successfully detected, enriched, and purified using FACS.
- Three distinct green fluorescent proteins were isolated.
- Two novel proteins exhibited 2.5-fold greater brightness than EGFP and possessed a unique cysteine-containing chromophore.
Conclusions:
- FACS offers significant advantages for screening low-frequency genes in bacterial cDNA libraries.
- This strategy enables the isolation of fluorescent proteins with enhanced properties from diverse organisms.
- The approach is adaptable for identifying non-fluorescent proteins using fluorescent indicators.