One-step, highly efficient site-directed mutagenesis by toxic protein selection.
A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plasmid. Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers. The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdB gene to inactivate the CcdB protein. The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids. Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%-100% was reached even after one round of transformation.
Xu
W
W
Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Zhang
Y
Y
Yeh
L Y
LY
Ruprecht
C R
CR
Wong-Staal
F
F
McFadden
B A
BA
Reddy
T R
TR
Ruprecht
R M
RM
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics* / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D019302:HIV Envelope Protein gp160 / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016601:RNA-Binding Proteins / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D004268:DNA-Binding Proteins; D019302:HIV Envelope Protein gp160; C489559:KHDRBS1 protein, human; D016601:RNA-Binding Proteins