Directed Evolution of Fluorescent Proteins in Bacteria.
Sara Mattson1, Geraldine N Tran1, Erik A Rodriguez2
1Department of Chemistry, The George Washington University, Washington, DC, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 15, 2022
Summary
Scientists evolved a novel far-red fluorescent protein in E. coli using directed evolution. This new protein, small ultra-red fluorescent protein (smURFP), is as bright as enhanced green fluorescent protein (EGFP).
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Directed evolution enables the creation of novel biomolecules.
- Error-prone PCR is a common method for introducing mutations.
- Fluorescent proteins are valuable tools in biological research.
Purpose of the Study:
- To develop a novel method for directed evolution of a far-red fluorescent protein in E. coli.
- To engineer a brighter and more efficiently expressed fluorescent protein.
- To adapt existing protocols for evolving other biomolecules in bacteria.
Main Methods:
- Utilized a novel method for directed evolution in E. coli.
- Employed genes to produce fluorophores internally within E. coli.
- Swapped the native phycocyanobilin fluorophore for biliverdin.
Main Results:
- Achieved a blueshift in fluorescence, enhancing quantum yield and brightness.
- Evolved a far-red fluorescent protein, named small ultra-red fluorescent protein (smURFP).
- smURFP demonstrated biophysical brightness comparable to enhanced green fluorescent protein (EGFP) and high expression levels.
Conclusions:
- The developed method successfully evolved a highly fluorescent and stable far-red protein in E. coli.
- The protocol is adaptable for directed evolution of other biomolecules in bacterial systems.
- smURFP represents a significant advancement in fluorescent protein technology for biological imaging.
Keywords:
Bacterial evolutionDirected evolutionError-prone PCRFluorescent proteinProtein engineeringRandom mutagenesisSynthetic biologysmURFPMore Related Videos
10:31Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability
Published on: February 3, 2022
3.0K
08:46Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
33.0K
