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Fig. 7 | Microbial Cell Factories

Fig. 7

From: Construction and development of an auto-regulatory gene expression system in Bacillus subtilis

Fig. 7

The schematic diagram of the construction of the E. coli–B. subtilis recombinant shuttle plasmids. a Fragment 1, harboring the ampicillin resistance gene (bla) and replication origin (pBR322 ori), was amplified from pUC19. Fragment 2, containing the replication protein (Rep), kanamycin resistance gene (neo r), and bleomycin resistance gene (ble), was similarly obtained from pMA09 by PCR. The terminus of each of the two fragments was flanked by a 30-bp homology corresponding to each other. b These two fragments were fused using the Sequence and Ligation Independent Cloning (SLIC) method [35], with some modifications, and yielding Plasmid pBSG01. c Construction of pBSG02 with the insertion of promoter PsrfA into pBSG01. d GFP was inserted downstream of the PsrfA, generating expression plasmid pBSG03. To compare the transcriptional strength, promoter PHpaII was substituted for PsrfA, generating plasmid pBSG04. To engineer PsfrA, the −10 (TAAACT) and −35 (GTGATA) motifs in PsrfA were replaced by the highly conserved sequence −10 (TATAAT) and −35 (TTGACT) region. The resulting plasmid was designated as pBSG05. The mutant promoter PsrfA was denoted as mutPsrfA

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