FMRG: Bio: Enabling Cell-Free Engineering and Biomanufacturing of Bacteriophages as a Universal Platform for Tailorable Bioactive Materials

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Purified protein synthesis machinery can create proteins outside the cell. Cell-free synthesis (CFS) can produce proteins that would otherwise disrupt or kill the cell that produced it. Vaccines have been produced using CFS. Synthesizing more complex structures, like bacteriophages, remains to be investigated and accomplished. Phages are a resource of a variety of bioactive materials. Their natural function is to infect specific bacteria. They could be reprogrammed to kill bacteria resistant to antibiotics. They could kill food-borne pathogens and treat bacterial infections, to name two important applications. The objective of this project is to develop the CFS technology required to produce phages flexibly and with high fidelity. Educational efforts involving the Cold Spring Harbor Laboratory DNA Learning Center will focus on developing and delivering curricular material. Topics critical to integrating CFS of phages into the biomanufacturing ecosystem will be emphasized. Developed for high school and college students, these materials will bolster the development of an agile bioeconomy workforce.The CFS of phages has not been exploited commercially. Basic research advances are needed that will enable the cell-free biomanufacturing of phages. The project will deliver knowledge on phage synthesis across species and on phage genomes. This fundamental knowledge will guide the engineering of phages with broader host ranges, by developing a one-pot next generation sequencing (NGS)-based approach for determining gene essentiality in phage and identification of permissive loci. The objective of the project is to lay the foundation for the cell-free engineering and biomanufacturing of phages, the largest family of antimicrobials. Efforts will address four limitations to the current production of phages: (i) the need for bioreactors of dangerous pathogens such as Salmonella or Listeria to produce; (ii) the likelihood of generating phage-resistant pathogenic strains; (iii) the need for many purification steps, which drives up the cost of phage production; and (iv) bottlenecks surrounding large DNA transformation into cells to produce the phage. The tools and knowledge developed can be readily used to tackle societal problems such as the emergence of antibiotic-resistant microbes. The phages included in the project are related to the pathogens Salmonella and Listeria monocytogenes (Lmo), two of the most significant pathogens in the U.S. To explore alternative solutions, several E. coli CFS systems will be optimized as effective, safe platforms for phage synthesis, complementary to a Gram-positive CFS system prepared from pathogen substitutes for Lmo.This Future Manufacturing award was supported by the Chemical, Bioengineering, Environmental and Transport Systems Division (ENG/CBET), the Division of Engineering Education and Centers (ENG/EEC), the Office of the Assistant Director (ENG/OAD), and the Division of Undergraduate Education (EHR/DUE).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.