[Supercopy plasmid based on the replicon from the temperate N15 bacteriophage]

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Mini-plasmids are crucial tools in molecular biology and biotechnology.
• The N15 temperate bacteriophage replicon provides a basis for novel plasmid development.
• High-copy-number plasmids are desirable for applications requiring significant DNA yields.

Purpose of the Study:

• To describe novel mini-plasmids derived from the N15 bacteriophage replicon.
• To characterize the N15-203 linear plasmid's high copy number and its utility as a cloning vector.
• To evaluate the transformation efficiency and cloning capacity of the N15-203 plasmid.

Main Methods:

• Construction of mini-plasmids based on the N15 bacteriophage replicon.
• Determination of plasmid copy number in lysogenic bacterial strains.
• Transformation efficiency assays using E. coli C (N15) strain.
• Insertion of foreign DNA into the N15-203 plasmid's BgIII cloning site.

Main Results:

• The N15-203 linear plasmid exhibits an anomalously high copy number (>250 per chromosome), constituting up to 50% of total cellular DNA.
• High copy number is dependent on lysogeny for the N15 phage and is lost in circular deletion variants.
• Transformation efficiency of N15-203 is comparable to pUC4K, and insertion of up to 20 kb DNA does not significantly reduce transformation efficiency or total plasmid DNA yield.

Conclusions:

• The N15-203 plasmid is a promising vector for molecular cloning of large DNA fragments.
• Its high copy number makes it suitable for biotechnological processes where vector productivity is critical.
• This novel plasmid system expands the toolkit for genetic engineering and synthetic biology applications.