(174bo) Optimization of Cell-Free Bacteriophage Synthesis of Bacteriophage T7

Clinical interest in bacteriophage as an antimicrobial agent has had a resurgence in recent years due to an increase of antimicrobial resistant (AMR) infections, which caused 1.27 million deaths worldwide in 2019. However, bacteriophage can infect and kill a limited number of bacteria strains, commonly only one, leading to multiple technical challenges in their production. These challenges include identifying and isolating a phage for treatment takes an average of 171 days; propagation of a phage requires a live strain of bacteria, usually a pathogen; due to endotoxins present in the bacteria, there is an added isolation step; and lastly, short-term storage of bacteriophage can significantly reduce the titer originally produced.

One solution to these barriers is the use of cell free bacteriophage synthesis (CFBS). The cell free system is an in vitro method that uses transcription/translation machinary from lysed bacteria, along with buffers, to mimic intracellular conditions. It is then used to produce a product, a protein (cell free protein synthesis) or a bacteriophage, from added DNA/RNA eliminating the need for live hosts. CFBS has been demonstrated to produce a handful of bacteriophages, including T7, in the past. However, CFBS has not been fully optimized for phage production. Only specific components of the cell free system have been optimized in the past, including the concentrations of DNA, magnesium-glutamate, potassium-glutamate, PEG-8000, and dNTP added to the reaction. In addition, the optimization methods previously utilized ignored potential interactions between the CFBS components.

I will use a design of experiment (DOE) multiple factor analysis method to more fully optimize the production of bacteriophage T7, to optimize more components and potential interactive effects. First, I will optimize factors influencing the cell lysate production: growth media, addition of 1 millimolar of magnesium to the growth media, sonication total energy, and length of the runoff reaction time. Second, I will optimize the factors directly involved in the cell free reaction: length of CFBS, and the concentrations of DNA, dNTPs, amino acids, magnesium-glutamate, potassium-glutamate, PEG-8000, DTT, and maltodextrin added. I will then combine the factors from both sets of data with the most impact of bacteriophage production to optimize the complete process.

Growth curves were completed using 2xYTP, 2xYTPG, and TB as growth media, with and without the supplementation of magnesium. TB had the highest overall growth, followed by 2xYTPG and 2xYTP. Magnesium supplementation further increased overall growth in each of the medias, with TB plus magnesium having the most growth. I hypothesize that lysates produced from TB and magnesium media will increase bacteriophage production, due to increased cellular machinery available in the media, which will be tested in the next set of experiments.