Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein |
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Authors: | Concha Nieto, Pilar Fern ndez de Palencia, Paloma L pez,Manuel Espinosa |
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Affiliation: | Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, E-28006, Madrid, Spain |
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Abstract: | We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible PM promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the PM promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the PM-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer. |
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Keywords: | pneumococcal malR gene plasmid pLS1 regulated promoters |
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