A bacteriophage is fundamentally a virus that exclusively targets bacteria. Whenever these phages target and lyse a bacteria, the clear area along with the remains of the lysed cells is known as a phage plaque. A simple way to understand is to imagine a tissue as a dense forest, with the cells being trees. A clearing in the forest, where there are no trees only some leaves and sticks on the group would be equivalent to a phe plaque. Due to the fundamental nature of bacteriophage lysis, the reproduced phages will always lyse outwards, developing several plaques. The main reason why plaques are so important is that we can harness and analyze the remains of the bacteriophage enzymes that carry out the lysing to understand the mutations of that phage, which was successful in lysing this one strain of bacteria.

Polymerase chain reaction (PCR) is a method used all over the world to multiply pieces of DNA from an initial strain. In our case, we can harness the power of PCR to screen and amplify the a screening and amplification matrix with the main goal of isolating the mutation from the phage and multiplying it so we can develop a large batch of phages that are successful in treating resistant bacteria.

The first step is to use PCR to isolate the mutation after harnessing and purifying the plaque. Plaque contains both wild-type phages and mutant phages, so both will be harnessed, however only the mutant phages will be put through the PCR multiplication process. Restriction enzymes can then be used to convert these mutations into specific point mutations, which is when the overall mutation can be translated down into the specific changes in each DNA nucleotide.

Now here is where the BRED system is used. The BRED system is especially important for recovering the pure mutations from the plaques, because it can analyze more than double compared to traditional Flanking primer PCR methods. This is why we are using a combination of PCR and BRED, to maximize the benefits and bypass the drawbacks of each. This model can still be automated because using BRED for plaque screening is still very-time consuming due to the very specific and sensitive nature of BRED. Our system has also shown that mutants which cannot be derived are not viable for real applications, however they can still act as a filter of sorts, reducing the time and effort required in analysis and sequencing.In terms of the success rate of the entire process, with current technology, mutant-containing phages can be recovered at an efficacy of anywhere between 3 - 22%, which will improve over time as the rate of technological developments increase. This percentage is for the recovery as well, future purification would still be needed to isolate the homogenous phage mutant.

The mutation would be developed through Phage Display for the mycobacteriophage and then used to construct gene deletions, replacements and heterologous gene insertions. This donor DNA contains the desired mutations flanked by homologous sequences of phage DNA to be engineered, which leads to homologous recombination occurring between the host phage genome and donor DNA. To make a deletion or small insertion, you will need to construct an approximately 200 bp double-stranded (dsDNA) substrate. For deletions, this should contain 100 base-pairs of homology upstream and downstream of the region to be deleted, making sure that that deletion will be inframe, if necessary. For small insertions, this substrate should contain the sequence to be inserted, flanked by about 100 base-pairs of homology on each end. To make a gene replacement mutant, you will need to construct a linear allelic exchange substrate (AES) that contains the sequence you wish to introduce, flanked by approximately 100 base-pairs of sequence homologous to either end of the region to be replaced. mutants containing one or more point mutations can be generated using two synthetic complementary oligonucleotides. All of the substrates are co-transformed into electrocompetent recombineering cells with the phage DNA, and plaques are screened for presence of mutation by PCR.

The problem with this system however is that it is very difficult in many cases to identify desirable bacteriophages that can actually lyse a bacteria when infected. We feel that phage-derived enzymes directly from virion-associated lysins, endolysin, and deploymerace can be used to lyse bacteria and can be used with any phage through intraperitoneal inoculation. The benefit of this is that they can lyse a wide range of species rather than strains. Several studies have exploited the fact that host range is linked to tail fiber composition for some phages. One scientist Yoichi genetically modified a T2 phage by swapping the long tail fiber genes (gp37 and gp38) with those from phage PP01, which specifically targets E. coli O157:H7. The exchange was done by homologous recombination between the genome of phage T2 and a plasmid carrying two regions of homology, flanking the gp37 and gp38 genes of PP01. As DNA synthesis, sequencing, and genome engineering tech will become more efficient, it will significantly expedite the possible host range.

We can also use phages for phagemids which encode plasmids to target certain resistance genes like for targeting the aph-3 kanamycin resistance gene that was packaged in the Staphylococcal phage ΦNM. Phasmids have been used to transfer foreign DNA across several bacterial species that helped express genes for protective antigens for a variety of pathogens.