SaPhARI Software

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1. We developed SaPhARI (Satellite Phage Algorithmic Recognition and Interpretation) a novel, versatile bioinformatics tool that streamlines the detection and classification of bacteriophage satellites, identifying both known and novel families in genomic and metagenomic data with precision and flexibility. This software exceeds the functionality of the only other software for satellite phage detection in multiple ways, particularly with its user definable and easily configurable parameters.

SaPhARI enhances the discovery process by identifying clusters of key proteins within genomic data, enabling researchers to efficiently analyze a wide variety of satellite families. With support for multiple input formats, including nucleotide FASTA and GenBank files, SaPhARI eliminates the need for cumbersome preprocessing, streamlining the analysis and making it accessible for both genomic and metagenomic research generated by the end user or accessed from sequence databases.

We harnessed SaPhARI to analyze over 30 metagenomes, successfully identifying 87 putative satellites in diverse environments such as human gut/stool, oral, and Antarctic marine samples. Our analysis of these protein clusters has revealed numerous putative novel satellite phage with limited similarity to characterized phages and provides significant insights into the distribution and diversity of satellite phages, demonstrating SaPhARI's capabilities. This work empowers researchers to uncover phage satellites in their own samples, and generates hypotheses for empirical testing, furthering our understanding of these important genetic elements in various ecological contexts. Even more significantly, by modifying parameters, this software can be utilized to discover any type of genomic element that an end user would select.

Along with SaPhARI we established a novel Bacteriophage Satellite Database that compiles information on 491 phage satellites spanning six distinct families, including phage inducible chromosomal islands (PICIs), capsid-forming PICIs (cf-PICIs), and the newly identified M. aichiense satellite family, "Phagelets." This comprehensive database not only serves as a critical reference for hypothesis testing but also supports experimental validation, providing transparent data that researchers can utilize in their studies. This serves as a resource for predictive modeling and as a resource for training sets.

Hardware: Colon & Soil Models

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2. We designed and built cost-accessible, reproducible hardware models that mimic natural conditions where circuits are likely to be deployed, specifically the human colon and soil environments. It is critical for synthetic biology to test systems in realistic environments where the constructs will ultimately be deployed; the colon and soil systems represent potential future applications in therapeutics and bioremediation.

Human colon model: Commonly used gastrointestinal simulators are very costly and difficult to produce and operate, presenting a significant roadblock for replication across labs with varying resources. To address this issue, we developed a cost-effective three-part colon model to test our engineered constructs in, and provided sufficient documentation to enable reproduction of the model which is a fraction of the price of commercial models. For the soil microcosms, we developed and documented large spatial soil microcosms which incorporated a rainfall simulator, nondestructive sampling, and plants. Not only have we designed, built, tested, and continually improved our hardware, but (as described below) we have also used both the colon model and the gut microcosms to test the ability of our engineered phage satellites to function in realistic environments. Our design, construction and implementation instructions, and associated data are publicly available.

Novel PDE Model

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3. We developed a group of first-of-their-kind partial differential equation models of phage satellite dynamics in natural environments informed by data collection from soil microcosms and model colon. Given the important role of satellite phages in regulating bacterial communities and their enormous potential for bioengineering and synthetic biology, it is essential to model the population dynamics of satellite phage in natural environments. Surprisingly, there is currently only a single publication that models satellite phages, one limited to the P2/P4 system (Mitarai, 2019). To address the need, we have written several partial differential equations (PDEs) which model the growth, fluid flow, and the infection dynamics of satellites.

Our project entailed the deployment of two types of phage satellites in the model colon and soil microcosms. The first type of satellite system is a P2/P4 system in E. coli with replicative and non-replicative variations of a kanamycin targeting CRISPR system, a negative control non-targeting CRISPR system, and a red fluorescent protein (RFP) satellite phage system. The second satellite phage system type is a Mycobacterium aichiense satellite phage. For each of these systems we wrote a PDE model which models the advection diffusion fluid dynamics, population dynamics, and Monod growth dynamics of each system. These models were essential in informing our experimental design, and delineating the behaviors of our satellite systems in a variety of natural environments.

These models demonstrated that satellite phages and transducing units persist and are infective in natural environments. Our model enhances the utility of satellite phage systems as vectors in synthetic biology and is a foundation for future modeling as satellites are better characterized and for future wet lab experiments as synthetic biologists use the potential of satellite phages in real-world environments.

Satellite Engineering

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4. We demonstrated the potential of E. coli P2-P4 phage satellite system by engineering it to perform a variety of valuable functions, including targeted killing of engineered bacteria, and reporting transduction with a red fluorescent protein in various engineered p4 cosmid constructs. Notably, we have tested these engineered constructs on two levels - both in vitro and "in situ," in our colon model and soil microcosms to ensure our constructs function in realistic environments. Both show promising results in vitro and in the colon and soil microcosms.

We engineered and employed the Kanamycin Resistance Targeting P4 cosmid to easily assay its sequence the P4 cosmid system’s sequence specific killing effect. We elected to use KanR as a target because it also functions as a useful proxy for an arbitrary toxin gene or virulence factor of clinical or scientific interest and also doubles as an antibiotic screening marker for our constructs. The construct showed an effective sequence specific killing effect in vitro and in our simulated colon. Given this success, we also engineered a dCas9 P4 cosmid vector used for targeted gene silencing (tested with success in vitro) and a dCas9-Omega construct fused to the omega subunit of RNA polymerase for gene upregulation (testing currently underway).

We also engineered a red fluorescent protein (RFP P4) construct to express a fluorescent phenotype to allow future users of the P4 cosmid system to assay the potency of P4 transducing agents. To do this, we replaced the Cas9 cassette in the cosmid with an RFP reporter device. This was tested both in vitro and in our simulated colon and soil microcosms where it showed promising results.

To find new phage satellite systems to engineer, we developed effective protocols for the discovery of novel bacteriophage satellites, and using this procedure we successfully discovered two novel, uncharacterized potential phages in E. coli from soil samples, a surprising and intriguing result given how well studied and characterized the E. coli is in the literature.

Novel Mycobacterium Satellite System

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5. We also pioneered a new phage satellite system in Mycobacterium, a genus selected for its environmental and clinical importance. This included discovery of novel Mycobacteriophage satellites, use of these satellites to overcome prophage immunity both in vitro and in our soil microcosms, and the engineering foundation for using satellites to expand host range of prophage.

We developed an effective protocol for physically isolating new satellite phage from environmental samples and for maintaining the titer of these novel satellite phage. Using our soil microcosm system, we demonstrated the ability of these satellite phages to persist in soil environments and to overcome prophage immunity and induce lysis both in vitro and in soil microcosms.

We engineered chimeric phage tail fibers predicted to expand host range of M. aichiense prophage, HerbertWM. In order to begin attempting to expand the host range of HerbertWM to other strains of Mycobacteria, we constructed two chimeric tail fiber constructs consisting of HerbertWM homologs as well as homologs of L5, another A2 phage. The phage L5 was chosen as the second component of the chimeric tail fibers after analysis of in silico predictive phage-host interaction which indicated this design would result in expanded host range of HerbertWM via the phagelets.

Education & Outreach

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6. In addition to numerous event introducing the public and the next generation of scientist to synthetic biology, we created comprehensive educational materials to bring phage satellites into the fold of synthetic biology: our comprehensive guide book of satellite-helper phage systems and our educational board game. These materials will help bring satellites to the forefront of synthetic biology with our comprehensive working knowledge of satellite systems and protocols for working with phage satellites. Our educational board game titled Gut Busters serves as an engaging and fun way to learn about phage satellites and their significance.