[The modern system for selection of cryptic plasmids based on replicon pMBI]

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The hybrid plasmid pGalI exhibits cytotoxicity linked to its genome integration site.
• This cytotoxic effect is influenced by flanking Eco RI recognition sites.

Purpose of the Study:

• To investigate the factors limiting the cytotoxicity of the hybrid plasmid pGalI.
• To explore the potential of other plasmids, such as pMB9 and those based on the pMBI replicon, in controlling pGalI cytotoxicity.
• To establish a method for cloning plasmids as cryptic plasmids by limiting pGalI cytotoxicity.

Main Methods:

• Analysis of the pGalI integration site and its association with cytotoxicity.
• Co-transformation experiments with pGalI and other plasmids (pMB9, pMBI-based plasmids) in E. coli.
• Selection of bacterial clones with a Gal+ phenotype to identify successful cryptic plasmid integration.

Main Results:

• Plasmid pGalI's cytotoxicity is associated with a specific genome site (1.1 mD) and Eco RI restriction sites.
• The cytotoxic effect of pGalI can be limited by co-transformation with plasmids like pMB9 or other pMBI-based plasmids.
• A reduction in the copy number of pGalI correlates with limited cytotoxicity.
• This limitation enabled the successful cloning of plasmids as cryptic plasmids in E. coli.

Conclusions:

• The cytotoxicity of hybrid plasmid pGalI is site-specific and can be modulated by other plasmids.
• Controlling pGalI copy number is key to mitigating its cytotoxic effects.
• This study provides a method for generating cryptic plasmids by leveraging pGalI's controlled cytotoxicity.