Abstract
In vivo
site-directed mutagenesis by ssDNA recombineering is a facile method to change the color of fluorescent proteins without cloning. Two different starting alleles of GFP were targeted for mutagenesis:
gfpmut3*
residing in the
E. 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 fluorescent protein. Eight different fluorescent proteins (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.
