pTCV-lac糞腸球菌穿梭質(zhì)粒

Schematic representations of plasmids pTCV-lac and pTCV-tet. (A) The components of the parental vector pTCV-lac are (i) the spoVG-lacZ translational gene fusion; (ii) the genes aphA3 and ermB, conferring resistance to kanamycin and erythromycin, respectively; (iii) the replication origins (oriR) of plasmids pACYC184 and pAMβ1; and (iv) the transfer origin (oriT) of the IncP plasmid RK2. Plasmid pTCV-tet was constructed by replacing the BamHI-PstI spoVG-lacZ-ermB fragment of pTCV-lac with a BamHI-PstI fragment containing the tet(M) gene of Tn916 devoid of its translational signals, including the first codon. The plasmid components are not drawn to scale. (B) Expression of the tet(M) reporter gene of pTCV-tet was obtained following cloning of EcoRI-BamHI fragments containing various tet(M) promoter regions to provide appropriate transcriptional and translational signals. MCS, multiple cloning site. (C) Expression of the spoVG-lacZ reporter gene of pTCV-lac was obtained following cloning of the same EcoRI-BamHI fragments as those for panel B to provide appropriate transcriptional signals. Note that in all the spoVG-lacZ fusions analyzed in this study, tet(M) translation ends prematurely at the underlined TAA stop codon and hence should not interfere with that of the downstream reporter gene.

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