Molecular Cloning of Segments of the M13 Genome

Abstract

The molecular cloning of individual genes of the bacteriophageM13 (Fig. 1) provides a new approach to the study of filamentous phage gene expression and provides the potential for complementation of a defective M13 phage. This paper describes the cloning of BamI-Eco RI* fragments of M13.¹ The cloning vehicle pBR317 was chosen for this study because it has been well characterized, can be obtained in high yield, and possesses two genes conferring antibiotic resistance on the host cell (Boyer et al. 1977). The cloning was carried out as described previously (Kaplan et al. 1977a). Two enzymes were used to digest the cloning vehicle and the M13 DNA, thus eliminating the possibility of intramolecular reactions. The plasmid pBR317 DNA was digested with Eco RI and Bam I; M13 RFI (superhelical, covalently closed, circular, duplex, replicative-form DNA) was digested with an excess of Bam I and a limiting amount of Eco RI*. Eighty Ap^r Tc^s clones were analyzed and 75 of them contained plasmid DNA ranging in molecular weight from 5.17 × 10⁶ daltons to 5.57 × 10⁶ daltons; the remaining five contained plasmid DNA of molecular weight greater than 5.57 × 10⁶ daltons. Two transformants, pKG7 and pKG35, were selected for further analysis.

RESULTS
Identification and Description of the M13 Inserts: Plasmid Molecular-weight Determinations
The two plasmid DNAs, pKG7 and pKG35, were electrophoresed on a horizontal 0.7% agarose gel (Kaplan et al. 1977b) along with plasmid DNAs of known molecular weights. The molecular weight of pKG7 was determined to be 5.60 ×...