Summary Phages are the natural viral predators of bacteria and are harmless to humans. The phages’ ability to recognize, bind, infect, and lyse host bacteria has led to their use as sensors for their hosts. Phages can be genetically engineered to express reporter proteins during the infection of their host, resulting in the release of the reporters following the lysis stage of infection. Our central hypothesis is that advanced methods in synthetic biology and genetic engineering will allow phages to be genetically optimized to perform as a dedicated nanobiosensor, and that phage-based assays can deliver genetic-level specificity of their host through reporter selection and optimization. The objective of this proposal is to use synthetic biology to abate current weaknesses identified for phage-based biosensing. The rationale for the proposed research is that while several weaknesses have been proposed for phage biosensors, advances in bioengineering and synthetic biology can provide solutions to address them. These issues include a lack of specificity within a species and a need for increased sensitivity. By mitigating these issues, phage-based sensors can be used to sensitively detect bacterial genetic sequences (e.g., pathogenicity and antibiotic resistance genes) with rapid results, low-cost, and minimal reagent storage conditions. Aim 1: Bioengineering phage infections for maximal reporter protein expression. Our working hypothesis is that the T4 infection conditions can be engineered to maximize the expression of reporter protein by optimizing lysis time and deleting genes non-essential for reporter expression on the phage genome. We will test the working hypothesis by engineering T4 phages with nonessential and structural protein genes deleted/silenced and complemented on a plasmid for propagation. Our expectation is a significant improvement in the limit of detection when used to detect wild type E. coli. Aim 2: Phage-enabled Recombinase Polymerase Amplification (RPA). Our working hypothesis, based on preliminary work, is that T4 phages bioengineered to have genes for RPA proteins can enable the genetic amplification of their host bacteria DNA following phage- induced lysis. To test the working hypothesis, we will genetically engineer the phage T4 with the genes for RPA- required proteins which will be expressed during infection of the bacterial host. Our expectation is that the post- infection lysate mixed with specific primers will allow detection of E. coli with genetic specificity. Following successful completion of the specific aims, the expected outcome is development of a suite of technologies which can bring phage-based biosensing across the technological “valley of death” and towards further application and commercialization. Phages, which allow rapid and low-cost detection of bacteria, suffer from a lack of specificity within a species. We will have demonstrated a method to significantly improve the expression of reporter proteins and a method to provide genetic level specificity while maintaining the overall benefits of phage-based detection (e.g., low-cost, rapid analysis, minimal reagent storage, bacterial lysis).

Bacteriophages genetically engineered to express the proteins needed for recombinase polymerase amplification (RPA) can enable genetic-level specificity of their host following lysis. Phages genomes can also be streamlined with the sole purpose of expressing high copies of reporter proteins during the infection of their host allowing better sensitivity and an overall reduced assay time. The advancements from this project will enable low-cost nanobiosensors which can concentrate bacteria from large samples, ultra-sensitively detect them with genetic precision and a low assay time while not needing refrigerated storage of reagents.