Team:NTNU Trondheim/Description

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Our project

A major challenge for phage therapy is the high host specificity of phages. A phage that is capable of killing one bacterial infection will not automatically be capable of killing another similar bacterial infection. In terms of safety and side effects, this is a good thing, as bacteriophages targeted towards harmful bacteria would not be capable of harming the cells or beneficial bacterial flora of the patient. However, in the case of a serious bacterial infection, it is obviously crucial for the patient to get the medicine in time to recover safely and well.

We were eager to try to counter this challenge. For our project, we therefore aimed to develop a method of evolving phages capable of infecting a targeted bacterial strain that they were previously not able to infect. This method needed to be able to quickly producing tailored phages, keeping us one step ahead of the resistant bacteria. Our project can be divided into 3 parts.

1) Collecting, isolating and purifying phages

Phages are plentiful in nature and exists wherever bacteria are found. By taking environmental samples from a wastewater treatment plant, we collected a broad range of bacteriophages. Bacteriophages capable of infecting and killing Escherichia coli DH5α were amplified by mixing the environmental water samples with growth medium and inocculating the mixture with E. coli DH5α.

Plaques of phages grown on Escherichia coli DH5α. First run.

The bacteriophages were purified by sentrifugation and filtering, and isolated by adding purified phage mix to growing E. coli DH5α and plating them on agar plates. The bacteriophages could be seen as plaques (empty spots with no bacterial growth) that was cut out of the agar and stored. In this way we got bacteriophages capable of infecting a given bacterial strain, E. coli DH5α.

See protocols for a detailed description of the isolation and purification process.

2) Evolving a bacteriophage capable of infecting target bacteria

In order to evolve bacteriophages capable of infecting target bacteria, we needed a controlled environment that favoured phages capable of infecting target bacteria. We built a bacterium/bacteriophage control system consisting of 3 coupled chemostats and two connected containers for medium (see figure). Photosensors capable of measuring real-time concentration of bacteria and phages in the out-flow were constructed. For a description of the hardware we built from scratch, see hardware.

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We had to set up a controlled environment that favoured phages capable of infecting some chosen target bacteria. In order to do this we first had to evolve phage host bacteria so that they could become resistant towards the phages. These were used as the target bacteria. The process was done in a single chemostat: When phages and bacteria are mixed, this creates an evolutionary pressure for the bacteria to develop resistance towards the phage, and those capable of developing this resistance will survive in the chemostat.

Once having one bacterial strain resistant towards the phage (target bacteria), and one bacterial strain non-resistant towards the phage (acting as phage hosts), we could proceed in the triple chemostat system (see figure):

Illustration of chemostat setup.

Medium from two separate containers was pumped into two separate chemostats, chemostat 1 and 2. Chemostat 1 contained the host bacteria (not phage resistant) and chemostat 2 contained the target bacteria (phage resistant). The contents of chemostats 1 and 2 were further pumped into chemostat 3 and mixed there. The bacteriophages were also pumped into chemostat 3. Phages, bacteria and medium was continously pumped out of chemostat 3 and measured to control the bacterial concentration.

The reason for this set-up was to be able to continously provide the bacteriophages with new hosts from chemostat 1, giving them a chance to evolve and overcome the resistance of the target bacteria pumped from chemostat 2. If not provided with available hosts, there was a risk that the bacteriophages would get flushed out of the chemostat because they didn’t evolve fast enough to be able to infect the target bacteria. With our set-up, this problem was avoided, so that the phages could continue infecting and replicating within the host bacteria and simultaneously evolving until they were also capable of infecting the resistant bacteria.

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3) Increase the mutation rate of bacteria

Since our platform relies on mutation/evolution of bacteria, it is useful to accelerate the natural mutation rate of the bacteria. We therefore contributed with a new biobrick, MP6, which is an inducible plasmid that can be transformed into E. coli and increase the mutation rate of the bacteria. The mutational activity is induced in the presence of Arabinose, and repressed in the presence of Glucose, making it possible to turn the transcription of the mutagenesis inducing proteins on and off. See parts and results for more details.

Email: igem-team@ntnu.no