M13 bacteriophage-based cloning vectors have been important biochemical tools in DNA sequencing, phage-displayed peptide libraries, and other applications. The first M13-based vector, M13mp1, was developed by Joachim Messing with the insertion of the lacZα gene in the ori. Variations followed in which restriction sites were incorporated to eventually create the M13mp18 polylinker. The insertion at position 5868 in the gene IV/II intergenic region happens to interrupt two binding sites for integration host factor (IHF), which is known to play a role in replication initiation. All M13mp phage are viable, but they have significantly lower propagation rates than wild-type M13 due to the replication defect caused by the insert. We aim to find a less sensitive location to place the lacZα gene. We chose positions 5927, 5620, and 5563 based in part on where inserts have been placed previously, but also in consideration of potentially sensitive features, such as the IHF binding sites, ori, gene IV transcript, and gene II promotor. We used PCR to amplify both the lacZα insert (from M13mp18) and the M13 backbones, each open at the appropriate position for the insert. Initial ligation attempts involved the assembly of fragments with overlapping overhangs, but these efforts have been unsuccessful. We shifted our focus to a more basic approach involving blunt-end ligation in a shotgun format to allow the phage to select the viable combination(s). We seek to learn whether it is possible to create an M13-lacZα phage that propagates faster than M13mp phage, perhaps even approaching wild-type M13.

Alternate locations for the lacZα insert in an M13-based cloning vector

2019 Spring National Meeting

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