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The Problem:
Circuit Control in Complex Natural Environments
In order to realize the promise of synthetic biology to bioremediate soil, regenerate and detoxify polluted seas, and deploy smart therapeutics, chassis hosting engineered circuits must be able execute their functions in a predictable, controlled manner in extremely complex, dynamic, and unpredictable environments.
This poses a major problem for synthetic biologists: the pressing need to develop novel tools and strategies to control, and leverage, the interactions of the synthetic circuitry with the crowded, inhospitable natural environment in which these circuits must function. This natural environment could range from a dysbiotic human gut or degraded soil in an agricultural field.
Our Solution:
SaPh:IRES (Satellite Phages: Integrated Real-World Engineering Solutions)
Harnessing the Exceptional Engineering Potential of Phage Satellite Systems
In order to address this challenge, the William & Mary iGEM team is pursuing a foundational advance project that exploits the treasure trove of synthetic biology parts and the profound wealth of unique tools offered by phage satellite systems.
Satellite phage or phage satellites–used interchangeably in the literature–are a diverse class of mobile genetic elements that parasitize other "helper" phage in order to propagate themselves. While the first satellite-helper system (the P2/P4 system) was discovered over fifty years ago, it has only been recently that a growing body of literature revealed the widespread prevalence, and diversity, of these systems (Six et al., 1973; de Sousa et al., 2023). Bacteriophage satellites are incredibly abundant in natural environments–marine phage satellites alone have a predicted abundance of 3.2 x 1026 (Eppley et al., 2022). The genetic mechanisms by which satellite phage parasitize their helper phage are likewise diverse and they have been hypothesized to have larger host ranges (Ibarra-Chávez et al., 2021). While it is likely that many more categories of satellite phages will be discovered, currently there are four distinctly classified types: P4-like, phage-inducible chromosomal islands(PICIs), capsid-forming PICIs(cf-PICIs), and PICI-like elements(PLEs) (de Sousa et al., 2023).
1. Satellite Phage Discovery
Relatively few satellite systems have been experimentally confirmed compared to their phage counterparts (Comeau et al., 2008). Currently, there is no comprehensive centralized resource available for satellite phages. The genetic diversity and lack of specificity of bacteriophage satellite elements presents further challenges to research efforts. These challenges hinder the ability of future synthetic biologists to study and engineer these systems.
a. Bioinformatic discovery of novel phage satellites: SaPhARI
To overcome these limitations, our project developed a comprehensive satellite database and an advanced bioinformatics tool. We have developed a novel software tool for synthetic biologists to identify phage satellites within genomic and metagenomic data. SaPhARI (Satellite Phage Algorithmic Recognition and Interpretation) leverages recent advances in metagenomic classification and statistical analysis. We have created a pipeline capable of identifying both known families of bacteriophage satellites and discovered new ones within genomic datasets. This approach harnesses modern software capabilities to enhance the characterization and understanding of phage satellites, particularly by identifying clusters of protein-coding genes within bacterial genomes as well as metagenome de novo assembled contigs. We also have developed a searchable, content-rich database of satellites.
b. Physical discovery of novel satellite systems
We developed screening strategies for new satellite systems in environmental samples in three key model systems, M. smegmatis, M. aichiense, and E. coli, using lysogenic bacteria, a concept which was discouraged in traditional screening methods. However, using bacterial lysogens allows us to not only screen for novel phages but also systems with helper phage and satellite dynamics. Satellite screening in these systems expands our understanding of these systems beyond purely computational predictions.
2. Applications of Satellite Phage
To address the challenges of deploying synthetic circuits in natural environments, SaPh:IREs leverages properties of satellites in three foundational applications: (a) delivering genetic circuits to manipulate bacterial gene expression at the population level, as well as (b) expanding phage host range, and (c) using satellites to overcome prophage immunity, the latter two essential for developing fieldable transduction. However, highly controlled laboratory conditions often fail to represent the more unpredictable and dynamic environments.
Our project therefore demonstrates and develops the profound potential of phage satellite systems for SynBio by determining behavior at different levels of complexity by testing the functionality of our phage satellite applications at three levels:
- In silico: Using modeling approaches to identifying and characterizing the behavior of satellite systems.
- In vitro: Engineering select satellite systems to address specific challenges in synthetic biology to demonstrate their potential for providing novel parts, devices, and systems.
- In situ: Testing our satellite systems in simulated natural environments, including a colon model and a soil microcosm model.
a. In Silico Modeling
Modeling Satellite Phage Dynamics and Predicting Host Range
Our project utilizes a first of its kind PDE (partial differential equation) model of satellite dynamics, which simulates satellites and the phages they parasitize by employing a series of differential equations. This model captures how these elements interact and develop over time in response to environmental factors. There are no models of this type which incorporate both realistic environmental dynamics and the wide variety of satellite phages. This modeling approach provides a comprehensive framework to predict and optimize the performance of phage satellites across different environmental conditions. These models informed our experimental approach which, in turn, improved the accuracy of the model.
We also developed an AI software to predict the interaction between phage and bacteria. The program assesses the phage genome and determines whether its host range is compatible with the strain of bacteria, which helped us select systems of satellite, helper phage, and target bacteria to use in our application, as well as assisting in designing novel mechanisms of expanding host range.
b. In Vitro Testing of Engineered Constructs
In order to convey the breadth of possibilities that each phage satellite system has to offer, we are using two largely different systems. The P2 helper phage and P4 satellite system in E. coli was the first to be discovered and has been the most characterized thus far. In contrast, the “phagelet” satellite in a strain of bacteria called Mycobacterium aichiense is extremely novel and little is known about its specific function and mechanisms. Although the uses of phage satellites are endless, our project focuses on three specific applications of them.
i. Sequence-Specific Population Level Control
One of the most unique qualities of phage satellites is its packaging mechanism. With relatively small genomes that lack many of the required genes to replicate, they utilize helper phage genes to construct a phage particle. Researchers in recent years have harnessed that characteristic by creating transducing units with the P2/P4 satellite system to be used as vectors for gene delivery (Fa-arun et al., 2023). Our team built off of these transducing units, which use a CRISPR cas9 cassette as a sequence-specific antimicrobial by creating several variations of the preexisting system. This includes new target sites for cas9 as well as replacing it with a dcas9 cassette used for gene silencing. We also replaced the CRISPR cassette with an red fluorescent protein (RFP) as a stand-in for future work with genes useful for bioremediation. For these versions we are making both replicable and unreplicable options to increase efficiency in the real world environment.
ii. Overcoming Prophage Immunity
To increase the fitness of the bacterial host, prophage often convey immunity to infection by other phages. However, with the emergence of antibiotic resistance, prophage immunity is creating increasingly more difficulties for phage therapy (Dedrick et al., 2021).
Mycobacterium aichiense, a strain of bacteria similar to Mycobacterium smegmatis, is known to express strong immunity to phage infection (Jacobs-Sera et al., 2012). This is due to the presence of the prophage HerbertWM which conveys a strong anti-phage defense mechanism. The “phagelet” is able to bypass this immunity by infecting M. aichiense and triggering the packaging and lysis of HerbertWM, ultimately killing the bacteria host. These “phagelets,” unfortunately, seem to be sensitive to degradation and were lost years ago. We developed a protocol for resurrecting denatured lysate and grew these “phagelets” to a high titer, clearing plates and killing M. aichiense in vitro.
iii. Increasing Host Range
Narrow phage host range has been decelerating innovations in phage therapy. We are combining engineering in the lab with machine-learning techniques to modify existing phage genomes, ultimately manipulating and expanding the host range through tail fiber engineering.
We are using the “phagelet” satellite system in Mycobacterium aichiense to increase the host range of the helper phage, HerbertWM. Although HerbertWM belongs to a family of phages known to infect M. smeg, the host range of HerbertWM does not extend beyond M. aichiense. We designed chimeric tail fibers for the prophage that will be assembled as the “phagelets” trigger the packaging of HerbertWM.
c. In Situ Experiment: Testing Constructs in Simulated Real-World Environments
Our engineering design includes testing these proof-of-concept systems in vitro, in semi-simulated environments, and finally in simulated in vivo conditions, which we refer to as in situ. The model colon is a three-stage continuous culture simulation of the human colon, representing the fluid and community dynamics of the colon environment. The soil microcosms model the colony structure of natural soil. The P2/P4 system was tested in a human colon model and both the P2/P4 and the “phagelet” systems were tested in soil microcosms to demonstrate the efficacy of our engineered system in real-world environments.
Inspiration
The inspiration for our project was threefold:
1) In 2018, the William and Mary iGEM team, as part of their outreach, screened for phages in M. aichiense with local high school students. As a result, they discovered a series of novel phage satellites which were called “phagelets”–the only satellites discovered in mycobacteria to this day. These satellites were thought to be lost shortly after being sequenced due to their fragile nature and they were left highly uncharacterized. When our 2024 iGEM team heard about these “phagelets,” we became determined to discover if they could be of use for synthetic biology.
2) As our team scoured the research that has been done on phage satellites when developing our project idea, we were struck by the growing body of literature that reported the widespread abundance of phage satellites in the virosphere, both phylogenetically and in sheer numbers (de Sousa et al., 2023).
3) We were also struck how underexplored these satellite systems were in the realm of bioengineering and synthetic biology. Satellites are diverse, in helper phage and bacterial host, in mechanism of infection, and in functions they can perform. Because of this diversity, we realized that satellites have immense engineering potential to better control biological systems. In particular, our team was very impressed and motivated by the Ibarra-Chávez et al. paper that described the potential of satellites for synthetic biology.