Engineering a phage to increase its host range is one of the fundamental barriers to widespread adoption of novel phage treatments. This is especially true for the nature of phages as for each strain of a species, there is one if not a few hundred different variants of the phage. Although this serves several benefits in directed, personalized and targeted medicine, the case to be made when it comes to treating infections often comes down to multiple bacteria at the root of the problem. That makes creating cocktails of tens of hundreds of different naturally occurring phages not just time consuming and expensive, but also ineffective at treating the disease.

This is where the idea of genetically modifying the target binding regions of a phage’s tail came to be, where we can just create 1 phage that could now expand its host-range through its targeting mechanism. Now, you can formulate not just custom but scalable phages that can treat all strains of E.coli, or perhaps both E.coli and TB and so on. The only problem is that the most accurate methods out there are CRISPR-Cas9 and other genetic insertion/deletion methods which are just as costly and hard to scale as traditional phage cocktails.

Thankfully, we can exploit the natural mechanisms of phages and bacteria for our own benefit: mutations. Over years, bacteria have developed advanced genetic responses to invaders and threats that can trigger mutations through its resistome. It’s what makes antibiotic resistance such a challenge, but this is also why phages are able to fight back. While antibiotics are static chemical compounds which do not evolve or change in response to AMR, phages are the biological competitor to bacteria and have, albeit different but effective, expansive genetic triggers that can allow them to mutate and circumvent the resistance mechanisms of bacteria. This not only makes resistance less of a factor for phage therapy because the rate at which phages mutate and change is faster than that of the bacteria, but it also presents an opportunity for the phages to directly counteract the resistance mechanisms that are mutated (i.e. phages can better attack efflux pumps).

Through phage mutation however, we can selectively force mutations in phages to attack multiple kinds of strains through homologous recombineering. The system comprises injecting DNA into specific points of the host’s genome or location. This can be used for altering the binding receptors of a phage to increase its range. However, traditional recombineering methods are ineffective at this because the phages cannot use selective markers such as drug-resistance genes for targeting the bacteria during viral lytic growth. Thanks to mutagenesis through homologous recombineering, this problem has been met but current systems are slow, unable to be used for all kinds of phages, and are not great at identifying useful mutations in the phages over several generations.

This is where BRED comes in: BRED or Bacteriophage Recombineering of Electroporated DNA is a highly efficient recombineering system that primarily uses electroporated phage DNA. Electroporation is when you apply an electrical field to an organism and make its cell membrane more permeable for insertion of DNA, drugs, and chemicals. In BRED, this means that mutants can easily be detected by PCR over the DNA (easily visible DNA) and it expedites the process significantly. BRED can be used to construct new, unmarked gene deletions, in-frame internal deletions, base substitutions, gene replacements with precision, and introduce gene tags for sequence identification.

Some techniques mediated by recombination proteins were used for E.coli using the lambda phage Red proteins, EXO AND BETA -> they then exploited Rac prophage for mutant construction, meaning that lytic growth is low and the role of antibiotic resistance pressure cannot be effectively used for mutant selection. Another key benefit is that thanks to the co-electroplating of both the phage DNA and targeting substrates, it becomes easier to engineer the substrate and its insertion, and when the mutations are then recovered, you can get a high proportion of copies that possess these mutations

Some techniques mediated by recombination proteins were used for E.coli using the lambda phage Red proteins, EXO AND BETA -> they then exploited Rac prophage for mutant construction, meaning that lytic growth is low and the role of antibiotic resistance pressure cannot be effectively used for mutant selection. Another key benefit is that thanks to the co-electroplating of both the phage DNA and targeting substrates, it becomes easier to engineer the substrate and its insertion, and when the mutations are then recovered, you can get a high proportion of copies that possess these mutations.

The recovery methods for these mutations are similar to most pipelines: the plaque that is formed through the phage replication and spread + lysis will be purified and analyzed against a wild-type (naturally occurring mutation) through PCR. When analyzing the selective mutant and wild type, you can quickly observe where the point mutations have occurred and depending on how easily the mutated types can be extracted, the more viable they will be for phage engineering. BRED can automatically expedite the process of identifying more useful and viable phage mutants as the process can recover 2-4x more pure mutations per plaque.

In fact, mutant-containing plaques can be recovered at an efficacy of 3.4-22.2%. After initial PCR screening of plaques, future purification is still needed to isolate homogenous phage mutants. Once the mutants are isolated, they will be then generated by infecting bacterial cells with the wild-type phage which would let the process to scale, following which extensive screening of recombinants will be required so that we can then identify the correct antibody for delivery using phage display.