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Fluorescent protein engineering by in vivo site‐directed mutagenesis
Author(s) -
Valledor Melvys,
Hu Qinghua,
Schiller Paul,
Myers Richard S.
Publication year - 2012
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1002/iub.1041
Subject(s) - recombineering , green fluorescent protein , mutagenesis , site directed mutagenesis , plasmid , escherichia coli , microbiology and biotechnology , shuttle vector , protein engineering , biology , genome engineering , chemistry , mutant , biochemistry , recombinant dna , genome , genome editing , dna , gene , vector (molecular biology) , enzyme
In vivo site‐directed mutagenesis by single‐stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site‐directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. © 2012 IUBMB IUBMB Life, 64(8): 684–689, 2012