Summary
Phages, which are the naturally evolved predators of bacteria, may hold the key to combating bacterial
pathogens, including the looming threat of multidrug resistant bacteria. Phages are viruses which while harmless
to humans and have been successfully engineered as tools to separate, concentrate, and detect their bacterial
hosts. Additionally, phages have been used as therapeutic agents to treat patients infected with pathogens
resistant to known antibiotics. While the potential benefits of phages are numerous, certain limitations must be
addressed in order to fully employ them. The central hypothesis of this proposal is that both top-down and
bottom-up approaches can be utilized to design and synthesize novel phages, through a combination of synthetic
biology and machine learning. This will result in phage-based tools with increased functionality and customizable
host ranges. The rationale for the proposed research is that as the threat of bacterial infections including those
with multi-drug resistance continues to grow, phages, which have evolved to efficiently recognize and kill
bacteria, will become indispensable tools. Therefore, the ability to rapidly design and engineer new phages for
biosensing and therapeutics will be a critical advantage to human health. The proposal contains three specific
aims which are supported by preliminary data and cited literature. Aim 1: Site-directed conjugation for advanced
phage-based biosensors and therapeutics. Under this aim, phages will be modified with alkyne-containing
unnatural amino acids allowing their direct conjugation to 1) azide decorated magnetic nanoparticles, and 2)
azide terminated polyethylene glycol. The modifications will allow the development of magnetic phages for
bacteria separation and detection, and phages that are more effective therapeutics due to their ability to avoid a
patient’s innate immune response, respectively. Aim 2: Decoding phage biorecognition elements using machine
learning. In this aim, machine learning will be used to model the binding of phages and their bacterial hosts. The
model will enable the prediction of host interactions as well as allow the design and synthesis of novel phage tail
fibers which can target specific bacterial isolates. Aim 3: Repurposing phage biorecognition for a broader host
ranges. Under the final aim, phage-binding proteins will be replaced with those known to recognize conserved
regions of the bacterial LPS, resulting in a phage with a much broader host range. This approach is innovative
because it uses top-down characterizations for bottom-up design and synthesis of novel phages. Traditional
phage screening methods will be replaced with the rapid synthesis of phages, which are optimized for a particular
bacterial isolate. Following the successful completion of the specific aims, the expected outcome is the design
and synthesis of phages that can be used to target a selected group of bacteria within Enterobacteriaceae for
advanced biosensing and therapeutics. A publically available computer model will allow rapid design of custom
phage biorecognition elements which can be added to functionalized phages. These technologies will allow
researchers to tip the scales of the co-evolutionary arms race between phage and bacteria.