Mutagenesis of T7 RNA polymerase

Abstract

For structural analysis of the phage T7 RNA polymerase, the phagemid pKU 01 carrying plasmids the T7 RNA polymerase gene under the control of lac promoter was constructed from pAR1219 and pUC119. Using recently developed, efficient random mutagenesis method of Xiube Liao, the C-terminal region of the T7 RNA polymerase was mutagenized. For a more efficient mutagenesis, a higher concentration of misincorporating nucleotide and longer misincorporation reaction time. As results a library of E. coli colonies containing mutations in the T7 RNA polymerase gene was constructed. Occurrance of mutations was suggested by the fact that colonies were formed when the mutated plasmids were inserted in E. coli BL21(DCAT4), which would not grow with wild-type T7 RNA polymerase gene.