An expression-packaging-processing vector which selects and maintains 7-kb DNA inserts in the blue T4 phage genome

Area of Science:

• Molecular Biology
• Virology
• Genetic Engineering

Background:

• Bacteriophage T4 is a versatile system for genetic manipulation.
• Efficient methods for inserting foreign DNA into T4 are crucial for research.
• The T4 ipIII gene encodes internal protein IPIII, involved in capsid packaging.

Purpose of the Study:

• To develop an efficient positive-selection vector for inserting foreign DNA into the bacteriophage T4 genome.
• To demonstrate the capacity for incorporating large DNA inserts (>7.0 kb) into the T4 genome.
• To establish a system for expression, packaging, and processing of fusion proteins within T4 phage.

Main Methods:

• Development of a positive-selection vector utilizing partial deletions of the T4 e gene.
• Insertion of foreign DNA segments fused to the T4 ipIII gene.
• Utilizing lysozyme activity for selection of plasmid integrants.
• Characterization of recombinant T4 genomes lacking the alt gene.

Main Results:

• Successful insertion of foreign DNA segments (>7.0 kb) into the T4 genome.
• Recombinant T4 phages express fusion genes driven by T4 ipIII promoters.
• Fusion proteins are packaged into the T4 capsid via IPIII targeting.
• Active IPIII::beta Gal fusion reporter protein is produced and packaged during phage infection.

Conclusions:

• The developed vector provides an efficient method for genetic manipulation of bacteriophage T4.
• The system allows for large DNA insertions and functional expression of fusion proteins.
• This expression-packaging-processing system is valuable for reporter protein studies in phage biology.