Advancing bacteriophage-based microbial diagnostics with synthetic biology.

Figure 1. Engineered bacteriophages can be assembled into sensitive, specific, and rapid microbial diagnostics. (A) Bacteriophages can be designed to express reporter payloads during specific microbial infection, leading to the production of detectable outputs. (B) Designing synthetic bacteriophages as microbial Synthetic biology is an emerging engineering field focused on designing artificial biological systems with novel functionalities for use in therapeutics, basic science, biotechnology, and diagnostics [1,2]. Continuous advancements in DNA synthesis and sequencing technologies coupled with new techniques for genomic modification and assembly have opened the door for harnessing the power and diversity of biology for applications. For example, natural bacteriophage products, such as ListShield (Intralytix) and Agriphage (Omnilytics), are commercially available for reducing unwanted bacterial contamination. Natural bacteriophages can be genetically modified to deliver engineered payloads into bacteria, thus selectively functionalizing target bacterial populations to produce active biomolecules. This strategy can endow bacteriophages with the ability to efficiently destroy bacterial biofilms or increase the bactericidal efficacy of antibiotics used in combination with phages by many orders of magnitude [3]. In addition, bacteriophages can be engineered as nearreal-time microbial diagnostics by using them to transform target bacteria into factories for detectable molecules (Figure 1A) [4]. Near-real-time microbial diagnostics are needed for food, clinical, industrial, and other environmental settings. The current state of the art for microbial diagnostics in food and environmental settings requires an enrichment step during which the pathogen of interest is selectively amplified over incubation times that can range from 10 to 24 h or more. The need for enrichment is to achieve adequate sensitivity and specificity, especially when dealing with complex samples. Although modern diagnostics can be sensitive and specific in laboratory settings, these techniques generally do not perform well in a heterogeneous mixture of competing microbes and environmental inhibitors without enrichment. These methods include traditional plating assays and antibody-based assays. Thus, in the absence of enrichment, most current tests are not sufficiently sensitive to detect a single or a small number of target microbes. By contrast, techniques such as PCR and hybridization-based assays may be sensitive but do not differentiate between living cells and dead cells, and require an enrichment step for specificity. In addition to being time consuming, enrichment necessarily increases the number of pathogens, a source of concern in food, clinical, and industrial settings, leading