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

纯化的蛋白质合成机器可以在细胞外产生蛋白质。无细胞合成（CFS）可以产生蛋白质，否则这些蛋白质会破坏或杀死产生蛋白质的细胞。合成更复杂的结构，如噬菌体，仍有待研究和完成。噬菌体是多种生物活性材料的来源。它们的自然功能是感染特定的细菌。它们可以被重新编程以杀死对抗生素有抗药性的细菌。 它们可以杀死食源性病原体和治疗细菌感染，这是两个重要的应用。该项目的目标是开发CFS技术，以灵活和高保真地生产可再生能源。涉及冷泉港实验室DNA学习中心的教育工作将侧重于开发和提供课程材料。将强调将生物燃料电池的CFS整合到生物制造生态系统中的关键主题。这些材料是为高中生和大学生开发的，将促进灵活的生物经济劳动力的发展。 需要基础研究的进展，这将使无细胞生物制造的昆虫。该项目将提供跨物种噬菌体合成和噬菌体基因组的知识。这一基础知识将指导具有更广泛宿主范围的噬菌体工程化，通过开发一锅下一代测序（NGS）为基础的方法来确定噬菌体中的基因必要性和鉴定容许位点。该项目的目标是为最大的抗菌剂家族--抗菌素的无细胞工程和生物制造奠定基础。努力将解决目前生产细菌的四个限制：（i）需要危险病原体如沙门氏菌或李斯特菌的生物反应器来生产;（ii）产生噬菌体抗性致病菌株的可能性;（iii）需要许多纯化步骤，这提高了噬菌体生产的成本;以及（iv）围绕大DNA转化到细胞中以产生噬菌体的瓶颈。开发的工具和知识可以很容易地用于解决社会问题，例如抗药性微生物的出现。该项目包括的细菌与沙门氏菌和单核细胞增生李斯特菌（Lmo）病原体有关，这是美国最重要的两种病原体。大肠杆菌CFS系统将被优化为有效、安全的噬菌体合成平台，与用Lmo的病原体替代品制备的革兰氏阳性CFS系统互补。（ENG/CBET），工程教育和中心司（ENG/EEC），助理主任办公室（ENG/OAD），该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.