| Chapter title |
Directed Evolution of Fluorescent Proteins in Bacteria
|
|---|---|
| Chapter number | 4 |
| Book title |
Fluorescent Proteins
|
| Published in |
Methods in molecular biology, January 2023
|
| DOI | 10.1007/978-1-0716-2667-2_4 |
| Pubmed ID | |
| Book ISBNs |
978-1-07-162666-5, 978-1-07-162667-2
|
| Authors |
Mattson, Sara, Tran, Geraldine N., Rodriguez, Erik A., Sara Mattson, Geraldine N. Tran, Erik A. Rodriguez |
| Abstract |
Directed evolution has revolutionized the way scientists create new biomolecules not found in nature. Error-prone polymerase chain reaction (PCR) introduces random mutations and was used to evolve jellyfish and coral fluorescent proteins in bacteria. We describe a novel method for the directed evolution of a far-red fluorescent protein in E. coli. The new method used genes to produce fluorophores inside E. coli and allowed changing the native fluorophore, phycocyanobilin, for a second small-molecule fluorophore, biliverdin. The directed evolution blueshifted the fluorescence, which enhanced the quantum yield to produce a brighter fluorescent protein. Finally, the evolution selected fluorescent proteins that expressed in large quantities in E. coli. The evolved fluorescent protein was named the small ultra-red fluorescent protein (smURFP) and was biophysically as bright as the enhanced green fluorescent protein (EGFP). This chapter describes the materials and methods used to evolve a far-red fluorescent protein in bacteria. While the focus is a fluorescent protein, the protocol is adaptable for the evolution of other biomolecules in bacteria when using a proper selection strategy. |
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Mendeley readers
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|---|---|---|
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