Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter.
By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196-198, TAT at 199-201, and ACC at 502-504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation.
Kitamura
Yoshihito
Y
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, 153-8904 Tokyo, Japan.
Mori
Satoshi
S
Chen
Wen
W
Sumaoka
Jun
J
Komiyama
Makoto
M
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D002563:Cerium / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D003062:Codon; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D049408:Luminescent Agents / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D002614:Chelating Agents; D003062:Codon; D049408:Luminescent Agents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D002563:Cerium; D004247:DNA; D004492:Edetic Acid; D011088:DNA Ligases