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[The modern system for selection of cryptic plasmids based on replicon pMBI]
Abstract:
Cytotoxicity of hybrid plasmide pGalI was associated with the genome site measuring for 1.1.mD and flanked by the recognizing sites of Eco RI restrictase. The cytotoxic effect of pGalI might be limited not only by plasmide pMB9 but also by other plasmides constructed on the base of pMBI replicone. Limitation of the cytotoxicity occurred due to a decrease in amount of the pGalI copies. Capacity of the plasmides studied to limit the pGalI cytotoxicity enabled to clone them as cryptic plasmides under conditions of both simultaneous with pGalI transformation of E. coli and the subsequent selection of clones with the Gal+ phenotype.
Insights
The hybrid plasmid pGalI
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.