A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis

Area of Science:

• Molecular Biology
• Microbiology
• Genetic Engineering

Background:

• Developing versatile cloning vectors is crucial for interspecies genetic manipulation.
• Shuttle vectors capable of replicating in both Gram-negative (e.g., E. coli) and Gram-positive bacteria (e.g., Streptococcus species) are highly valuable.

Purpose of the Study:

• To construct and characterize a novel bifunctional shuttle vector, pVA838, for use in both Escherichia coli and Streptococcus sanguis.
• To demonstrate the utility of pVA838 for cloning streptococcal DNA fragments in E. coli and subsequent transfer to S. sanguis.

Main Methods:

• Construction of the pVA838 shuttle vector by in vitro ligation of pACYC184 and pVA749 replicons.
• Characterization of pVA838 replication, marker expression (chloramphenicol resistance, erythromycin resistance), and unique restriction enzyme sites.
• Utilizing pVA838 as a shuttle vector to clone streptococcal plasmid fragments in E. coli and transform them into S. sanguis.

Main Results:

• The 9.2 kb pVA838 plasmid replicates and expresses erythromycin resistance in both E. coli and S. sanguis.
• Chloramphenicol resistance is expressed only in E. coli; insertion inactivation is possible at EcoRI or PvuII sites.
• pVA838 contains unique SalI, SphI, BamHI, NruI, and XbaI sites for molecular cloning and can be amplified in E. coli but not S. sanguis.

Conclusions:

• pVA838 serves as an effective shuttle vector for cloning and manipulating DNA between E. coli and S. sanguis.
• The vector's features, including selectable markers and unique cloning sites, facilitate genetic studies in streptococci via E. coli intermediates.