Team:Munich/Design

Phactory

Project Design

First Blick of Phactory

The Problem

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. Antimicrobial resistance is on the rise and the death toll from bacterial infections will continue to increase if alternative solutions are not found. 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 antibiotics. An alternative – phage therapy – has an extensive history of application in countries with limited access to antibiotics. The overarching problem our project therefore needed to solve is that although phage therapy has been known for almost a century, there is still no widespread application of phage therapy.

In researching the hurdles impeding phage therapy, we identified the production of phages as one of the most striking problems. Specifically, the current production process is inefficient, leads to a lot of impurities and contamination, requires the cultivation of human pathogens in large quantities, and causes regulatory problems due to imprecise manufacturing standards and a lack of adequate quality controls.

We ideated ways to use synthetic biology to overcome these challenges and found that using cell extract as the central component of our manufacturing pipeline might allow us to overcome these issues. With the central piece of our project in place, we could then break down the overall problem into isolated, solvable sub-problems we could work on in parallel. We created design modules for these sub-problems, 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 did not impede progress in another module. We defined quantitative criteria to measure progress in achieving our design 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.

The Solution: Phactory

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. Furthermore, the presence of DNases complicates the production of phages from linear genomes. This module therefore had two separate goals:

The first goal was to produce our own, affordable cell extract and to establish quality control mechanisms to quantify its performance. [lyophilization at some point]

The second goal was to optimize cell extract for the expression from linear DNA.

[…]

Synthetic Phage Manufacturing

Our cell-extract is capable of host-independent phage assembly from a linear DNA template. Attempting to generate maximal phage titers, we decided to screen for incubation time, influence of dNTP addition and optimal DNA template amount.

To determine the verifiability of our phage product, we chose to combine three different methods. A plaque assay is a cheap and simple way to assure the quantify functional phages. Reproduction capability of our phages was tested with a burst size analysis and the transcription capacity by reverse transcriptase qPCR.

Modular Phage Composition

When thinking about a release into the environment in the course of a therapeutic application, it is important to avoid having genetically engineered phages. We postulated that in our cell-free system it should be possible to modify phage proteins without altering their genome. To test our hypothesis, we chose to externally express an edited HOC (highly immunogenic outer capsid) protein and incorporate it in the assembly reaction. Considering the isolation of the desired protein out of a heterogeneous solution, we chose to add a histidine-tagged protein containing YFP or NanoLuc luciferase for a possible application in medical imaging.

Quality Control

Clinical applications require highly pure samples free of bacterial endotoxins and contaminations and with highly functional bacteriophages. 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 show 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 Nanopore sequencing. Nanopore sequencing was chosen because of compatibility with our additional goals: At a current cost of 1000€ for a starter kit it is easily the sequencing technology with the lowest investment cost. Its long read lengths allows for unambiguous identification of the source of contaminations. Being small enough to fit into a pocket, the device is highly portable.

As a submodule we decided to construct a software suite called Sequ-Into that allows anyone to easily determine the amount of contaminants in the first few minutes of sequencing, thereby making this aspect of phage manufacturing accessible without specialized knowledge in bioinformatics.

The purity of our samples, quantified using a LAL-test, was sufficient for oral administration, while the endotoxin levels were too high for direct intravenous application. However, oral delivery suffers from rapid degradation of the phages in the acidic gastric juice.

Lacking access to expensive packaging or purification machines, we decided to build two 3D-printed low-cost devices to overcome these hurdles independently. The hardware was designed to be compatible with existing laboratory equipment or open source solutions.

Verifying the Functionality of Phages

Verifying Toxin Levels

For intravenous administration, our approach was to purify the bacteriophages from the remaining cell-extract via fractionation in a pressure-driven size-exclusion filter system. Phage survival is quantified via a plaque assay. To examine their suitability for intravenous application, additional LAL-test could be performed.

Encapsulation

For oral application, we built a nozzle to encapsulate the phages in monodisperse Calcium-alginate microspheres protecting them in the stomach. We quantify the size and monodispersity of the alginate capsules using brightfield and Epifluorescence microscopy. Survival and release of phages in simulated gastric and intestinal fluid, respectively, is assessed via plaque assays.

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.