New shuttle vector for cloning in Bacillus stearothermophilus

Area of Science:

• Molecular Biology
• Microbiology
• Biotechnology

Background:

• Development of novel cloning vectors is crucial for advancing genetic engineering and synthetic biology.
• Existing vectors may have limitations in host range, stability, or transformation efficiency.

Purpose of the Study:

• To construct and characterize a new cloning vector, plasmid pRP9, with enhanced properties for gene cloning applications.
• To evaluate the transformation efficiency and stability of pRP9 in various bacterial hosts.

Main Methods:

• Plasmid construction based on the broad host-range plasmid pLM6.
• Transformation assays in Bacillus subtilis, Bacillus stearothermophilus, and Escherichia coli.
• Segregational and structural stability tests under selective pressure.

Main Results:

• Plasmid pRP9 (2.9 kb) was successfully constructed and demonstrated efficient transformation in B. subtilis, B. stearothermophilus, and E. coli.
• pRP9 exhibited very high segregational stability in Bacillus hosts.
• High structural stability of pRP9 carrying a 3-kb fragment was observed in Bacillus strains under selective conditions.
• No aberrant DNA forms (single-stranded or high-molecular weight) of pRP9 were detected in B. stearothermophilus.

Conclusions:

• Plasmid pRP9 is a small, stable, and efficiently transformable cloning vector suitable for use in B. subtilis, B. stearothermophilus, and E. coli.
• The developed host/vector systems possess the necessary properties for efficient gene cloning.
• pRP9 represents a valuable tool for molecular biology research and biotechnological applications.