Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Area of Science:

• Molecular Biology
• Virology
• Genetic Engineering

Background:

• Bacillus subtilis phage phi 1 possesses a genome with limited restriction endonuclease cleavage sites, making it a candidate for gene-cloning vectors.
• Initial characterization revealed two phage variants, phi 1E1 and phi 1E2, with one and two EcoRI cleavage sites, respectively.

Purpose of the Study:

• To develop Bacillus subtilis phage phi 1 into a functional gene-cloning vector.
• To create a deletion mutant of phi 1E2 (phi 1E2 delta 1) to expand the range of DNA insert sizes.
• To assess the utility of the engineered phage for cloning DNA fragments generated by various restriction enzymes.

Main Methods:

• Induction of a deletion mutant (phi 1E2 delta 1) from phi 1E2.
• In vitro recombination experiments using phage rho 11 DNA.
• Analysis of cloned DNA fragments using restriction endonuclease digestion (EcoRI, BamHI, HaeIII).

Main Results:

• The deletion mutant phi 1E2 delta 1 successfully cloned EcoRI-digested fragments of varying sizes.
• Analysis of ten phi 1 clones revealed that six contained rho 11 DNA fragments inserted at EcoRI sites.
• Hybrid phage DNAs were cleaved by BamHI and HaeIII, indicating successful insertion of foreign DNA recognized by these enzymes, unlike the parental phage DNA.

Conclusions:

• The engineered Bacillus subtilis phage phi 1E2 delta 1 serves as a viable vector for cloning DNA fragments generated by EcoRI digestion.
• The resulting hybrid phages demonstrate utility as vectors for cloning foreign DNA fragments produced by BamHI or HaeIII digestion.
• This research expands the toolkit for genetic manipulation using bacteriophages.