| Title |
Fluorescent protein engineering by in vivo site‐directed mutagenesis
|
|---|---|
| Published in |
IUBMB Life, May 2012
|
| DOI | 10.1002/iub.1041 |
| Pubmed ID | |
| Authors | |
| Abstract |
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. |
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X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United States | 1 | 50% |
| Australia | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
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| Scientists | 1 | 50% |
Mendeley demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
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| United Kingdom | 1 | 3% |
| Belgium | 1 | 3% |
| Unknown | 27 | 90% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 8 | 27% |
| Student > Master | 6 | 20% |
| Student > Bachelor | 3 | 10% |
| Researcher | 3 | 10% |
| Student > Postgraduate | 3 | 10% |
| Other | 3 | 10% |
| Unknown | 4 | 13% |
| Readers by discipline | Count | As % |
|---|---|---|
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| Biochemistry, Genetics and Molecular Biology | 9 | 30% |
| Immunology and Microbiology | 1 | 3% |
| Chemistry | 1 | 3% |
| Engineering | 1 | 3% |
| Other | 0 | 0% |
| Unknown | 4 | 13% |