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Título: | Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae |
Autor: | Ruiz-Masó, José A. CSIC ORCID ; López-Aguilar, Celeste CSIC ORCID; Nieto, Concha ; Sanz, Marta CSIC; Burón, Patricia CSIC; Espinosa, Manuel CSIC ORCID ; Solar, Gloria del CSIC ORCID | Palabras clave: | Plasmid pMV158 Cloning vectors Regulated expression Pneumococcal malR gene |
Fecha de publicación: | ene-2012 | Editor: | Elsevier | Citación: | Plasmid 67(1):53-59(2012) | Resumen: | We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter PM of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from PM is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive Ptet promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae | Descripción: | 18 p.-4 fig. | Versión del editor: | http://dx.doi.org/10.1016/j.plasmid.2011.09.001 | URI: | http://hdl.handle.net/10261/61079 | DOI: | 10.1016/j.plasmid.2011.09.001 | ISSN: | 0147-619X | E-ISSN: | 1095-9890 |
Aparece en las colecciones: | (CIB) Artículos |
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PLASMID_G. del Solar_2012_postprint.pdf | 259,94 kB | Adobe PDF | Visualizar/Abrir |
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