The Problem
Antimicrobial resistance is on the rise and the death toll from bacterial infections will continue to increase if no alternative solutions are developed. The threat of antimicrobial resistance has been known for a long time - Alexander Fleming, the discoverer of penicillin, predicted in 1945 that “exposing […] microbes to nonlethal quantities of the drug makes them resistant”. Even though efforts for development of novel antimicrobial compounds have been stepped up in recent years, there is still a lack of safe and effective substitutes for antibiotics1. A promising alternative – phage therapy – has an extensive history of successful application in countries with limited access to antibiotics. Although phage therapy has been known for almost a century, widespread application of phage therapy still has to become reality.2 This was the overarching problem our project needed to tackle.
In order to design an impactful project for this year’s iGEM season, we deemed it important to precisely understand and define the problem we wanted to work on.
Through extensive research into the factors impeding phage therapy, we identified the production process to be one of the most striking problems. In particular, the current methods are inefficient, lead to high impurities and contamination, require the cultivation of human pathogens in large quantities and causes regulatory problems due to imprecise manufacturing standards and a lack of adequate quality controls.
The Solution: Phactory
We contemplated ways to use synthetic biology to overcome these challenges and found that using cell extract as the central component of a manufacturing pipeline for phages might allow us to overcome these issues. With the central piece of our project in place, we were able to define the individual modules of our manufacturing pipeline. The modular approach allowed us to break down the overall problem into several isolated, solvable sub-problems that could be worked on in parallel. For each module we defined the requirements we wanted to fulfill and brainstormed potential solutions for reaching these requirements. The individual modules were designed in such a way that they are truly independent of one another and that issues in one module would not impede progress in another module. We defined quantitative criteria to measure progress in achieving the identified requirements during the build-test cycle. To maximize our chances of success and the robustness of our designs, we chose the simplest solutions we could come up with. We defined additional goals when multiple solutions fulfilling these criteria were available: accessibility, portability, affordability, and safety.
Phactory was designed to be an accessible manufacturing pipeline that produces pure, precisely defined bacteriophages at medically relevant concentrations using highly portable, affordable and modular components.
Preparation of Cell Extract
Commercially available cell extract is highly expensive, while home-made alternatives are time-consuming to produce and lack reliability. Furthermore, the presence of DNases in cell extract complicates the production of phages from linear genomes. This module therefore had two separate goals:
The first goal was to produce high-quality, affordable cell extract and to establish quality control mechanisms to quantify its performance. The preparation protocols described in previous publications were analysed to identify optimization goals. 3 To achieve these goals every step of the preparation process was assessed and improved, with a particular focus on cell cultivation and cell lysis. To enable easy distribution and storage, we decided to improve the overall durability and storage time of our cell extract by lyophilization. After resuspension of the dried cell extract, assembly of any given bacteriophage can be performed.
The second goal was to optimize cell extract for the expression from linear DNA. To accomplish that, we identified the intracellular DNAse RecBCD as our main target. This enzyme - in cooperation with RecA - is an essential component maintaining the integrity of the bacterial genome4. Furthermore it is the protein causing degradation of linear DNA within cell extract. Therefore, the inactivation of this endonuclease was defined as a crucial step to ensure efficient phage production within the cell extract.
Synthetic Phage Manufacturing
Our goal was to use cell extract as a host-independent platform for phage assembly from a linear DNA template. To demonstrate the universal applicability of our manufacturing platform, we attempted to express a variety of structurally different phages at titers suitable for therapeutic application. In order to generate maximal phage titers, we identified the parameters that potentially impact phage production. These included incubation time, influence of dNTP addition and optimal DNA template concentration.
Modular Phage Composition
When thinking about a release into the environment in the course of a therapeutic application, it is important to avoid using genetically engineered phages. We postulated that in our cell free system it should be possible to modify phages without altering their genome. In a modular approach, we aimed at adding both natural phage DNA and engineered proteins to an assembly reaction. To test our hypothesis, we chose to externally express an edited HOC (highly immunogenic outer capsid) protein and incorporate it in the assembly reaction. Thinking about the possible applications of fluorescent and bioluminescent phages in medical imaging, we designed expression constructs encoding for YFP or the NanoLuc luciferase. Additionally, we needed to consider first the isolation of the desired protein and then later the purification of the phages out of a heterogeneous solution. Therefore, we added a histidine-tag.
Quality Control
Clinical applications require highly pure samples of functional bacteriophages, free of bacterial endotoxins and contaminations. The purpose of this module is to implement quality control mechanisms to ensure a safe and effective application.
Quantifying the Level of DNA Contamination
While gels and absorption can be used to detect the presence of the desired phage genomes, they do not prove the absence of contaminating DNA. In fact, we found that more than 50% of isolated phage genome purified from common E. coli phages using traditional protocols is instead E. coli genomic DNA. To precisely quantify the amount of contamination, we used third generation sequencing. Nanopore sequencing was chosen because of its affordability and small size that makes the device highly portable. Its long-read lengths allows for unambiguous identification of the source of contaminations.
As a submodule, we decided to construct a software suite called Sequ-Into that allows the user to easily determine the amount of DNA contamination in the first few minutes of sequencing, thereby providing access to this aspect of phage manufacturing without requiring specialized knowledge in bioinformatics.
3D printed low-cost devices to overcome these challenges independently. The hardware was designed to be compatible with existing laboratory equipment or open source solutions.
Verifying the Function of the Phage Product
To assess the antimicrobial action of our phage product, we combined two methods. We chose a plaque assay as a cheap and simple way to confirm the presence of functional phages and to quantify the phage titer. Reproduction and transcriptional capability of our phages however could be assessed by reverse transcriptase qPCR.
Reducing Toxin Levels
In an attempt to push our purity towards enabling intravenous administration, we decided to perform fractionation in a pressure-driven size-exclusion filter system. Suitability of the phage product for intravenous application could then be assessed with an additional endotoxin test.
After these steps, our phages would be ready for animal testing, which we did not plan to attempt in the context of the iGEM competition
Encapsulation
The transport within the body often poses problems for the activity and integrity of phages, which can be addressed by packaging of the phages in a protective layer5. For oral application of our phage product, sufficient stability for gastric passage has to be guaranteed. The highly acidic environment, as well as the presence of proteases composed the major challenges. Our packaging method thus needed to provide resistance to low pH. At the same time, phages needed to be released from their protection upon reaching intestinal fluid. These characteristics are provided by calcium-alginate microspheres6. Lacking suitable encapsulation hardware, we decided to build our own nozzle to encapsulate the phages in monodisperse microcapsules.
The methods most suitable for quantification of size and assessment of monodispersity of the alginate capsules were brightfield and epifluorescence microscopy. To determine whether our encapsulation method fulfills our requirements of survival and release of phages in simulated gastric and intestinal fluid, respectively, we subsequently performed plaque assays.
References
- Incentivising innovation in antibiotic drug discovery and development: progress, challenges and next steps.
- A century of the phage: past, present and future.
- Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology.
- RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks.
- Malik, D. J., Sokolov, I. J., Vinner, G. K., Mancuso, F., Cinquerrui, S., Vladisavljevic, G. T., … Kirpichnikova, A. (2017). Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Advances in Colloid and Interface Science, 249, 100–133.
- Colom, J., Cano-Sarabia, M., Otero, J., Aríñez-Soriano, J., Cortés, P., Maspoch, D., & Llagostera, M. (2017). Microencapsulation with alginate/CaCO3: A strategy for improved phage therapy. Scientific Reports, 7, 41441.
