Proposed Implementation

End Users

The OhioState 2021 team initiated phage therapy utilizing genetically engineered bacteriophages. These phages will be injected into the patient and treat sepsis. The phage’s genetic information will be implanted into the bacteria to make a protein inhibiting lipid A molecules in bacteria, avoiding the immune system overreaction and eliminating sepsis. A phage database was developed with the goal of providing teams with information they need, or a place to start.

This year, our proposed users continue to be patients suffering from sepsis. However, this year new aspects were added to OhioState 2021 team’s project to help further address the problems of sepsis. One major issue with current sepsis treatment is that the primary technique for diagnosis, blood sample culturing, takes 48 to 72 hours. This method is also very expensive, making sepsis diagnosis inaccessible to countries with less robust healthcare systems.

To reduce this time window and costs, we developed a construct to be built into a phage for use. This construct contains a newly implemented strong promoter sequence along with the gene sequence for mCherry, a fluorescent protein, and we planned to clone it into Escherichia coli. This would theoretically cause the E. coli to glow very quickly because of the increased transcription rate of the strong promoter. Techniques like this could eventually replace or heavily supplement blood sample culturing for sepsis diagnosis. However, E. coli is not the only common sepsis-associated bacteria. Moving forward, our team would expand our product to encompass more bacteria and actually clone into phage.

NEXT STEP - Cloning Phages

Going forward, our team would have a few options for implementation: cloning this construct into different phages, or designing novel constructs for each type of sepsis-causing bacteria (for example, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Klebsiella pneumoniae, group B streptococci, etc).

Different phages will allow us to probe the bacteria sample until we find a phage that makes the sample fluoresce, identifying the bacteria type. Creating a different construct for each bacteria would mean still using different phage but each phage have a unique fluorescent gene with a specific color. In this way, different colors would correspond to the different bacteria types.

The easiest way to proceed will be to use different phages with the same construct. This would allow us to use a much simpler and less expensive testing method; which would only have to register the mCherry wavelength in order to diagnose patients with any common sepsis-causing-bacteria.

NEXT STEP - Phage cocktails for detection

Having decided on using only a singular construct, phages that infect different bacteria, specifically common sepsis-causing-bacteria, will be needed. Each phage that targets these bacteria will be incorporated into different phage cocktails. These cocktails will only have up to four different types of phage so that when a cocktail causes fluorescence, only four phage have to be tested to see which ones actually infected. In order to verify this process, a test runs can be conducted. A known bacteria can be inserted into a blood like sample and a phage selected from the database to infect that bacteria. Then, the phage can be added to the sample and fluorescence observed. A lack of fluorescence means that the concentration of phage added should be increased. If fluorescence is observed, then a new phage type can be added to the solution and retested to make sure a phage cocktail won't change a positive result.

If our diagnostic went to market, it would be used in a straight forward manner. When a patient’s blood sample is taken, it will be partitioned out and each of these phage cocktails applied to one partition. After a length of time that is to be determined, ideally around 2 hours or so, a UV light can be shined on each partition. If a glow is not observed for any partition, it is unlikely there is a gram-negative bacterial infection. If any partition glows, it signifies the presence of bacteria. The cocktail used to infect the glowing partition will now be split into its component phages and applied to a new uninfected partition. After waiting, any glow observed can now be directly related to the phage infecting it. Using the phage database, the bacteria can largely be identified, knowing the phage that infected it and the phage that didn’t infect it.

Once the bacteria is determined, the therapeutics side from last year can take over. Phages (including the phage that detected the bacteria) that infect the identified bacteria, again sourced from the database, would be engineered into their therapeutic, phighter phage form. These different phages will again be combined into a cocktail and administered to the patient.

NEXT STEP - Further developing the Bioreactor Model

With the further development of bacteriophages in wet lab this year, dry lab looked to the future to determine methods of mass producing bacteriophages. The team decided to set the foundation of the development of the bioreactors by creating a dynamic model for the phage concentration for the bioreactor. The dynamic model would be the foundation for the development of the bioreactor process control system. Process control allows for systems to account for deviations in different variables in the system and continue operating at the optimal conditions.

After developing the process control system, the next step would be to physically create the bioreactors and implement the control system. Our model would then be updated taking into the data that is measured during the experiment. The control system would now at this point incorporate both the dynamic model equations and data. This would lead to a more accurate model, and with further testing (if deemed necessary) would be ready to be used for use in production.

In terms of production, the bioreactor will largely be used for all types of phage production. Using engineered phage as “seeds”, mass amounts of engineered phinder and phighter phage can be produced for any specific needs. These bioreactors will be crucial to the implementation of our project which relies on phage creation and availability.