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Figure 2 | Microbial Cell Factories

Figure 2

From: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus

Figure 2

Construction of plasmids containing the epothilone biosynthetic gene cluster and their expressions in M. xanthus. a Diagram of the plasmid construction. The plasmid p15A-recTp and p15A-recMx8 contain the transposase gene (IR-Tpase-IR) and the site specific attachment element (Mx8), respectively. Homologous arms (epoup, epoCov, epodown) and selective genes (tet, apra, galk, sacB) were constructed into the plasmids. The construction of the plasmids p15A-TP-CF and p15A-Mx8-CF, carrying the entire epothilone gene cluster, were performed by two rounds of recombination. Firstly, the linearized Fosmid3B11 was electroporated into the E. coli, which recombined with p15A-recTp or p15A-recMx8 to generate the plasmid p15A-Tp-fos or p15A-Mx8-fos. Then, the linearized Cosmid10 was introduced for recombination with p15A-Tp-fos or p15A-Mx8-fos to create the p15A-TP-CF or p15A-Mx8-CF. b PCR amplification verification of constructs. Left panel shows the size indication of the plasmids p15A-TP-CF, p15A-Mx8-CF and Fosmid3B11. The middle and right panels are digestion analyses of p15A-Mx8-CF and p15A-TP-CF colonies by restriction enzymes KpnI, respectively. M1 and M2 are DNA markers. c PCR amplification verification of the presence of epothilone biosynthetic genes in M. xanthus recombinants. The cross marks in b and c indicate the false positive clones.

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