APES

Accelerated Phage Evolution System

Motivation

The ability of bacteria to develop resistance to antibiotics has been the underlying theme of the project. A large advantage phage therapy possesses over conventional therapy involving antibiotics is the ability of phages to adapt and overcome any resistance bacteria develop to them. However, this potential of evolution is not without its limits, as has been shown^[1] and any process that gives the phages a leg up in the arms race is a step forward. APES is intended to step in and save the day.

The idea was to use random mutagenesis to produce a phage library by increasing the mutation rate of the phages. The next idea was to automate the process utilising a hardware system, minimising the effort required.

System

The phage-bacterium pair chosen for this was E. coli strain MG1655 and Enterobacteria phage T4, since they are well characterised and easily available. Before building the hardware, the actual protocols to be followed for mutagenesis had to be designed and tested for efficiency. The procedure involved two steps: first, exposure of phages to mutagen, and second, screening for mutant phages with the ability to infect and lyse bacteria that have developed resistance to non-mutant phages. UV radiation was chosen as the mutagen, since it is cheap and easily available. Further, there were existing studies that studied the possibility of UV radiation being used as a mutagen.^[2]

The Process

To perform the screening procedure, phage resistant bacteria are obtained. For this, E. coli strain MG1655 is plated on a lawn flooded with a large amount of T4, (around 10⁸ pfu, sufficient to produce clear lawns in most platings) and incubated overnight. Each single colony formed is then picked and cultured to obtain stocks of phage resistant bacteria.

For the mutagenesis, the phages are taken in eppendorf tubes and irradiated with UV light (wavelength 298 nm) for varying durations. Prolonged mutagen exposure results in most of the phages being rendered non-viable due to fatal physiological or DNA damage, while lesser than optimal exposure times results in lower mutation efficiencies. Using these irradiated phages, plaque assays are to be performed with the resistant strains. If plaques are observed, then the mutant phages in the plaque are capable of infecting and lysing the bacteria which are resistant to wild-type strains of the phage. These plaques are picked up and plated on the resistant bacteria a second time for further screening.

Details

In all, 30 samples of resistant bacteria were obtained. 100 μL of 10⁸ pfu/mL of the phages in SM buffer were taken in eppendorf tubes and exposed to UV radiation by placing the tubes in a standard transluminator. The exposure was carried out for durations of 1 min, 2 min, 4 min, 8 min, 16 min, 32 min, 1 hr, 2 hr, 4 hr and 6 hr.

However, the first set of trials produced very few mutants capable of producing plaques when plated with the wild-type resistant phages, and also had a distinct lack of reproducibility. Before getting into more trials with UV, a chemical mutagen, EMS, was used with the same procedure. Again, no meaningful results could be obtained.

This process of mutagenesis was modelled using stochastic matrices. It was observed that, given a long enough time frame, either every possible strain of the genome becomes equally probable (and hence, contributes to a negligible fraction of the net virus population) or the process tends to a state where the original strain has a very low probability of mutating. This also explained the irreproducibility of the experimental results, as the process of mutagenesis was random. This model, combined with the lack of sufficient resources, motivated our team to discontinue APES as a wet lab endeavour.

Further description of the model can be found here.

References

1] Labrie, Simon J., Julie E. Samson, and Sylvain Moineau. "Bacteriophage resistance mechanisms." Nature Reviews Microbiology 8.5 (2010): 317."
2] Yarosh, Daniel Bruce. "UV-induced mutation in bacteriophage T4." Journal of virology 26.2 (1978): 265-271.