Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Site-specific recombination is crucial for bacteriophage lambda integration and excision.
• Hybrid plasmids offer a tool to study these genetic processes in vivo.
• Understanding these mechanisms is key for genetic engineering and biotechnology.

Purpose of the Study:

• To construct and analyze hybrid plasmids containing lambda genes for site-specific recombination.
• To investigate the role of Int protein in the integration of these plasmids into the Escherichia coli chromosome.
• To determine the conditions affecting plasmid integration and excision in different bacterial strains.

Main Methods:

• In vitro construction of hybrid plasmids carrying lambda recombination genes.
• Analysis of protein synthesis in E. coli minicells.
• Plasmid integration studies in wild-type and polA1 E. coli strains.
• Southern blotting to confirm chromosomal integration.

Main Results:

• Hybrid plasmids successfully integrated into the E. coli chromosome at the attB site.
• Constitutive expression of the int gene is necessary for Int protein synthesis and integration.
• Plasmid integration was observed even in polA1 strains, with genetic markers maintained for over 50 generations.
• High Int protein levels allowed for prophage excision independent of the Xis product.
• A six-fold decrease in Int protein concentration critically affected both integration and excision.

Conclusions:

• The int gene and attP site of lambda are sufficient for hybrid plasmid integration under conditions of effective Int protein expression.
• Hybrid plasmids can integrate into the bacterial chromosome without compromising cell viability.
• Int protein concentration is a critical factor regulating both integration and excision of lambda DNA.
• These findings provide insights into the molecular mechanisms of bacteriophage lambda site-specific recombination.