Project Summary
Bacteriophages, phages for short, are extremely abundant and diverse in the environment but
also very under sampled and understudied. A major factor limiting the characterization of more phages
is the lack of fast and efficient methods to do so. Alghough, techniques have been developed to find the
receptor of a given phage on its host cell, no techniques to find phages dependent on a given receptor
have been developed. However, this second method may be even more important. Such a technique
would allow a user to select a bacterial membrane protein or structure of interest and preferentially
select environmental phages that require it for infection. This could be useful for understanding the
fluctuations of bacterial membrane structures across different ecological systems or for informing which
environmental phages will make the best therapies.
This proposal will develop a novel and generalizable assay for receptor-guided discovery
of environmental phages using co-culture. The current standard technique includes a classic plaque
assay where a single host strain of bacteria is grown in a lawn with an environmental sample of
phages. Each phage infecting the host bacteria causes a clearing in the lawn called a plaque, and
unique phages are not identifiable based on the appearance of the resulting plaques. The novel assay
proposed here adds a new visual signal by co-culturing multiple strains of bacteria in the lawn. Each
strain is a single gene knockout and tagged with a unique fluorescent maker. Phages with infection
cycles independent of any of the proteins knocked out will lyse every strain on the plate and leave no
fluorescent signal in the plaque. Phages of interest will lyse only a subset of the bacterial strains in the
lawn, and the fluorescent signal corresponding to the required protein knockout will show through the
plaque. In this way the novel assay differentiates plaques by the dependencies of the original infecting
phage and allows for targeted isolation of phages of interest in just one experimental step. This project
will be completed in a highly interdisciplinary environment where the experimental protocols,
computational analysis, and hardware can all be developed with equal rigor.
This assay will be developed first in Escherichia coli as a model system and then generalized
into Pseudomonas aeruginosa to move towards clinical applications. Knockout proteins will be chosen
first to test the limits of the assay and later to search for interactions between phages and bacterial
virulence and antibiotic resistance factors. Taken together, this work will develop a novel assay for
targeted phage isolation that can be applied to ecological studies, single phage therapies, and
construction of phage cocktails.