Site-Specific Integration of the Double-Mutation Glucose Isomerase (GIG138PG247D) Gene in Streptomyces lividans and Its Stable Expression |
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Authors: | Yonghui Yang Chong Xu Feng Ge Zhiqiang Lu Guoping Zhu Hui Li Jun Liao Liwen Niu Yuzhen Wang Mian Wu |
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Institution: | (1) Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Chinese Academy of Sciences, Hefei, Anhui 230026, P. R. China, CN |
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Abstract: | A recombinant expression plasmid pYH12, containing the double-mutation glucose isomerase (GIG138PG247D, GI2) coding gene and
its natural regulatory sequence, was constructed for site-specific integration in Streptomyces. The resulting plasmid was introduced into Streptomyces lividans TK54 by protoplast transformation and two apramycin-resistance (AmR) transformants, designated GY2 and BY7, respectively, were obtained further based on enzyme assays. These results for polymerase
chain reaction (PCR), Dot blot, and recovery of cloned fragments from the transformant chromosome indicated that the GI2 gene
was integrated into the S. lividans chromosome by site-specific recombination, and which was further verified by Southern blot. We found that the free form of
plasmid pYH12 co-existing with the integrated form was present in S. lividans. SDS-PAGE analysis showed that the GI2 gene was expressed in S. lividans. The intracellular GI2 specific activity was 1.15 U/mg. The stability of integrants demonstrated that the cloned GI2 gene
was stably integrated and expressed even in the absence of selective pressure.
Received: 28 March 2001 / Accepted: 14 May 2001 |
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