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Updated: May 29, 2026
A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction
Published on: May 17, 2024
Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in
José A Ruiz-Masó1, Celeste López-Aguilar, Concha Nieto
1Molecular Microbiology and Infection Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E28040 Madrid, Spain.
Abstract:
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 P(M) of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P(M) 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 P(tet) 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.
Insights
We developed a new plasmid vector, pLS1ROM, for controlled gene expression in Streptococcus pneumoniae. This tool enables maltose-inducible expression of target genes with high regulation and stability.
Area of Science:
- Microbiology
- Molecular Biology
- Genetic Engineering
Background:
- Streptococcus pneumoniae requires robust tools for genetic manipulation.
- Regulated gene expression is crucial for studying bacterial physiology and pathogenesis.
- Existing vectors may lack stability or precise control for pneumococcal applications.
Purpose of the Study:
- To construct and validate a novel plasmid vector for inducible gene expression in Streptococcus pneumoniae.
- To engineer a system with high regulation and low background expression.
- To ensure the stability and utility of the vector for genetic studies.
Main Methods:
- Construction of the pLS1ROM plasmid vector based on the pMV158 rolling circle replication (RCR) plasmid.
- Incorporation of the maltose-inducible P(M) promoter from the malMP operon.
- Inclusion of the malR repressor gene in cis under a constitutive promoter for tight regulation.
- Cloning of the green fluorescent protein (GFP) reporter gene to test functionality.
Main Results:
- The pLS1ROM vector demonstrated successful maltose-dependent expression of the GFP reporter gene.
- GFP fluorescence was observed only in the presence of maltose, indicating effective induction.
- Undetectable background fluorescence was achieved in the absence of the inducer (maltose).
- The vector exhibited excellent structural and segregational stability in S. pneumoniae.
Conclusions:
- The pLS1ROM plasmid vector provides a reliable and highly regulated system for gene expression in Streptococcus pneumoniae.
- This vector facilitates genetic manipulation and the study of gene function in pneumococci.
- The maltose-inducible system offers precise control over gene expression, minimizing off-target effects.
