The M13-based vectors developed by Joachim Messing and coworkers have served as important tools in the preparation, cloning, and sequencing of single-stranded DNA. In the more mature versions, such as M13mp18, a polylinker is housed in the lacZα gene, which itself is embedded in the origin of replication of the phage. DNA fragments of interest can be conveniently inserted into the polylinker and then sequenced from a common primer annealed to the single-stranded genome. The original vector was constructed by ligating the lacZα gene to restriction fragments of the wild-type M13 genome. The successful clone, M13mp1, was found to contain the lacZα insert after position 5868 in the (+) strand origin. The descendants of M13mp1 are perfectly healthy phage, but we recently demonstrated that the rate of propagation of M13mp18 is significantly lower than that of wild-type M13. We suggested that the location of the insert between the two binding sites of integration host factor (IHF) may affect the activity of M13 protein II (pII) in replication initiation at the (+) strand origin. We seek to examine whether phage containing the insert in alternative locations could have propagation rates closer to that of wild-type. We used PCR to build lacZα inserts to be placed at positions 5563, 5620, and 5927 using DNA fragment assembly. In choosing these locations, we were mindful of potentially sensitive areas of the gene IV/II intergenic region, such as the pII and IHF binding sites, the (-) strand origin, the gene IV transcript, and the gene II promotor.

Construction of a more virulent lacZα-containing M13 phage vector

255th National Meeting (2018)

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