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bioRxiv · 10.1101/2023.03.02.530847

In Vitro Evolution to Increase the Titers of Difficult Bacteriophages: Rapid Appelmans Protocol

Kok, D. N.·Turnbull, J.·Takeuchi, N.·Tsourkas, P.·Hendrickson, H. L.
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Abstract

Bacteriophages are becoming increasingly important in the race to find alternatives to antibiotics. Unfortunately, bacteriophages that might otherwise be useful are sometimes discarded due to low titers making them unsuitable for downstream applications. Here, we present two distinct approaches to experimentally evolve novel New Zealand Paenibacillus larvae bacteriophages. The first approach uses the traditional agar-overlay method, whereas the other was a Rapid Appelmans Protocol (RAP) modelled after the established Appelmans Method. Both approaches resulted in an increase in plaque-forming units (PFU/mL). The RAP approach was significantly faster and simpler, and allowed us to evolve a set of bacteriophages in as little as four days, increasing titers 100-1000-fold relative to their ancestors. The resultant titers were sufficient to extract and sequence DNA from these bacteriophages. An analysis of these phage genomes is provided. We also propose a model that describes the parameters that allow the RAP approach to select improvement of bacteriophage titer. The RAP approach is an effective method for experimentally evolving previously intractable bacteriophages in a high-throughput and expeditious manner.

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Kok, D. N., Turnbull, J., Takeuchi, N., Tsourkas, P., Hendrickson, H. L.. 2023-03-02. In Vitro Evolution to Increase the Titers of Difficult Bacteriophages: Rapid Appelmans Protocol. https://doi.org/10.1101/2023.03.02.530847

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