Bacteriophage recombineering in the lytic state using the lambda red recombinases.
Bottom Line: Bacteriophages, the historic model organisms facilitating the initiation of molecular biology, are still important candidates of numerous useful or promising biotechnological applications.With the help of BRED, we removed a copy of mobile element IS1, shown to be active, from the genome of P1vir, a coliphage frequently used in genome engineering procedures.Overall, P1virdeltaIS provides a genome engineering vehicle free of IS contamination, and BRED is likely to serve as a generally applicable tool for engineering bacteriophage genomes in a wide range of taxa.
Affiliation: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary. firstname.lastname@example.orgShow MeSH
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Mentions: Phage P1virΔIS was grown on E. coli MG1655, and was titred as described in Experimental procedures. The mean phage titre was 1.16 × 1010 plaque forming units (pfu) ml−1, which falls in range of that of P1vir lysates, generated by the same protocol. Dependence of phage titre on multiplicity of infection (moi) yielded a similar tendency for both phages (Fig. S3). Moreover, based on plaque morphology, P1virΔIS is indistinguishable from P1vir (Fig. S4). The one‐step growth curves of P1virΔIS and P1vir are compared on Fig. 4. The latency time, rise period, and the burst size of the two phages are also basically identical (Fig. 4).
Affiliation: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary. email@example.com