Creation of circularly permutated yellow fluorescent proteins using fluorescence screening and a tandem fusion template.
By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong similarity to YFP and therefore can be used in replacement.
Chiang
Jason Jui-Hsuan
JJ
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, M5S 3G9, Toronto, Ont., Canada.
Li
Isaac
I
Truong
Kevin
K
D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria