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Step-by-Step Phage Production Breakdown
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01. Perfect Phage Engineering <span class="tb-accordian-toggle fa fa-angle-down"></span>
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<p>The first step is to find a method to increase the overall host range of the phage itself. We plan to carry this out through the use of targeted mutagenesis through the Bacteriophage Homologous Recombineering with Electroporated DNA (BRED) model. 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.</p>
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02. Development of Phage Antibody with Bacterial Antigens <span class="tb-accordian-toggle fa fa-angle-down"></span>
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<p>The second step of the overall solution and production breakdown is to develop the phage antibody through the use of bacterial antigens. This will be carried out through the phage display. We initially would start by packaging the antibody production genes inside a viral protein "coat". The antibody genes develop an antiboy "hat" which is attached on the outside of hte entire phage-coat structure which forms a phage antibody. That antibody "hat" functions like a real antibody via the binding to very specific antigens on a bacteria. By changing the antibody production genes, we can create multiple antibody "hats". We can leverage phage display and antibody libraries through this process. What would then happen is that we'd use a sample of the disease as bait for the antibodies and test the bacteria over the entire phage display library. Only the phage antibodies which target that specific bacterial strain will develop a strong attachment to it. We'd then replicate that specific antibody production gene and elimiante the others. We'd finally modify the coat protien which carries out the antibody sequence with the antigen from the bacteria. This can be derived from the hate since the hat only binds to a given bacterial strain.</p>
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03. Formation of the Transduction Particles <span class="tb-accordian-toggle fa fa-angle-down"></span>
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<p>After this, our goal now would be to create the genetic code to activate the immune response and replicate the transduction particles. This can be carried out through the inocolation of the antigen development sequence, the combination of that sequence with virion protein, followed by the addition of phage-derived enzymes for lyses. When discussing the potential of specifying the genetic transduction across engineered phages, it can be difficult to inhibit not just the bacterial resistance mechanisms when phages latch on but also start an immune response. However, a potential method can be used to produce transducing particles which can be used as helper phage-free particles as an antimicrobial agent that can go beyond lytic phage therapy. The assembly comprising virion proteins and the non bacteriophage nucleic acid cargo is called a transducing particle. IN preparation of transducing particles, structural proteins were applied by helper bacteriophages. Upon cell lysis, helper phage progeny and transducing particles are released, which can infect recipient cells, either continuing the helper bacteriophage lytic cycle or completing tradnuction. </p>
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04. Mass Production <span class="tb-accordian-toggle fa fa-angle-down"></span>
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<p>The main goal with mass production is to be able to convert the one-perfect phage into multiple for the use of oral distribution. This can be carried out through the host-reactor process. We use a specific system known as the CAVE system the help with this process. CAVE system is a foundation of mutagenesis that can be used for both host-range increasing and enhancing characteristics of bacteria. This is done through selective mutation which is discussed slightly above. We use evolution and mutations and pick the best mutations for optimizing bacteriophages. This can be used for almost all features of bacteriophages. For example, using mutagenesis can allow a phage to possess the binding receptor of OMKO1 which can infect Pseudomonas aeruginosa using the outer membrane porin M (OprM) which is part of the antibiotic efflux pump.</p>
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05. Vaccine Administration <span class="tb-accordian-toggle fa fa-angle-down"></span>
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<p>Bacteriophages can be used as a novel and efficient category of gene delivery vehicles (GDVs) for the introduction of various diagnostic and therapeutic cargoes to human cells. Using phages as delivery mechanisms are not toxic and pose no side effects. Bacteriophages inject their genetic material into bacteria by recognizing the lipopolysaccharides, pili, peptidoglycans, proteins and teichoic acids comprising the cell walls and outer membranes of the bacteria. Although vaccine administration through subcutaneous or intramuscular injection is more widespread than other administration routes, phage-based vaccines are stable in the gastrointestinal tract and thus represent potential to be used orally. (Oral vaccinations) Tell the patients to take probiotics.</p>
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<p>The main goal of the 6th step is to produce the antibodies against the pathogenic bacteria. Generally, APCs engulf the entire phage and our goal here is to extract the genetic devleopment sequence from the transduction particle. We would then process and present that to the T cells to the lymphocytes to produce the antibodies. The non-essential capsid proteins of phage T4 can be used to display 2 different foreign proteins, one an antigen-production cell (lysogen promoter) and another a Dendritic Cell Receptors. The expression of protein could be most-probably Fc receptor or a C-type lectin receptor, determined by major histocompatibility complex (MHC) and identifying which ones are viable MHC molecules to promote phagocytosis. Dendritic cell-associated promoters will be required to attach with the phages.</p>
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<p>The goal for the final step is to disable resistance mechanisms, lyse the bacteria, and reproduce the transduction particles. Here's how this would look like: Bacteriophage latch onto the cell and inject the transduction particle. The transduction particle will takes care of everything else, mainly focusing on lysing the bacteria as dicussed above (producing more copies of the phage while simultaneously killing it). </p>
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Gram-negative, rod-shaped bacterium, 61% mortality rate
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