Affiliation:
1. grid.258151.a 0000000107081323 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University 1800# Lihu Road 214122 Wuxi People’s Republic of China
2. OriGene Biotechnology Co., Ltd. 88# Meiliang Road 214122 Wuxi People’s Republic of China
3. State Key Laboratory of Dairy Biotechnology 1518# Jiangchang West Road 200436 Shanghai People’s Republic of China
Abstract
Abstract
The gene integration method is an important tool to stably express desirable genes in bacteria. To avoid heavy workload and cost, we constructed a rapid and efficient method for genome modification. This method depended on a mobilizable plasmid, which contains a P tac promoter, an introduced multiple cloning site (iMCS), and rrnBT1T2 terminator. Briefly, the mobilizable plasmid pK18-MBPMT with the P tac-iMCS-rrnBT1T2 cartridge derived from pK18mobsacB was prepared to directly integrate hetero-/homologous DNA into the Corynebacterium glutamicum genome. Like our previous method, this method was based on insertional inactivation and double-crossover homologous recombination, which simultaneously achieved gene overexpression and inactivation in the genome without the use of genetic markers. Compared to the previous method, this protocol omitted the construction of a recombinant expression plasmid and clone of the target gene(s) cassette, which significantly decreased the workload, cost, and operational time. Using this method, the heterologous gene amy and the homologous gene lysCT311I were successfully integrated into the C. glutamicum genome at alaT and avtA loci, respectively. Moreover, the operation time of this method was shorter than that of the previous method, especially for repeated integration. This method, which is based on the mobilizable plasmid pK18-MBPMT, thus represents a potentially attractive protocol for the integration of genes in the course of genetic modification of C. glutamicum.
Funder
Youth Program of Natural Science Foundation of Jiangsu Province
Youth Foundation of Jiangnan University
Publisher
Oxford University Press (OUP)
Subject
Applied Microbiology and Biotechnology,Biotechnology,Bioengineering
Cited by
2 articles.
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