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Cell Free Platforms for Prototyping and Biomanufacturing


There is a critical need for capabilities that will enable DoD to leverage the unique and powerful attributes of biology to solve challenges associated with production of new materials, novel capabilities, fuels, and medicines. This topic is focused on improving the utility of cell-free systems as a platform technology to address key technical hurdles associated with current practices in engineering biology. A successful platform should address several or all of the bottlenecks associated with the state-of-the-art in cell-free systems, including production of cell-free reagents with improved consistency and scalability, improved methods for characterizing and validating cell-free reagent preparations, new cell-free systems to expand the number of organisms capable of being modeled, and improved reproducibility of results over scaled volumes. In addition, these cell-free platforms should be distributable in a format that can be readily transitioned to academic, government, and commercial researchers, all of whom rely on the ability to rapidly assay engineered biological systems. Biological production platforms have great potential to provide new materials, capabilities, and manufacturing paradigms for the Department of Defense (DoD) and the Nation. However, the complete realization of this potential has been limited by current approaches to engineering biology that rely on ad hoc, laborious, and time-consuming processes, as well as the large amount of trial and error required to generate designs of even moderate complexity. One technology that could address many of these bottlenecks is the use of cell-free systems for the rapid prototyping and testing of biological systems. Conventional approaches to engineering genetic systems rely on molecular cloning into DNA vectors, transformation or transfection of cells, antibiotic resistance-based selection, growth in appropriate media, and assaying cells for the desired function. While significant progress has been made toward improving these processes, engineering living cells is inherently costly, slow, and complex. By short circuiting many of the steps required for in vivo gene expression, cell-free systems offer several advantages that could potentially transform the state-of-the-art, including reduced cost, increased throughput, decreased system complexity, and the ability to be utilized in a distributed setting. In addition, cell-free systems enable the production and testing of cytotoxic compounds, the prototyping of pathways with toxic metabolic intermediates, and for the production of molecules, such as proteins containing non-standard amino acids, that are difficult to engineer into living systems. Although the use of cell-free assays has significant potential to rapidly engineer and test biological systems, several technical hurdles remain that have prevented widespread adoption of the technology. Methods for preparing reagents used in cell-free experiments are often inconsistent, which can lead to irreproducible results. In addition, current methods do not produce batches of a sufficient volume of high quality reagent to enable widespread use. Furthermore, existing internal controls are insufficient for the complete characterization and validation of reagents, which makes instituting process controls difficult. The cell-free platform itself also requires improvement, as only relatively simple biological processes have been demonstrated and in only a handful of organismal environments. PHASE I: Develop an initial design and determine the technical feasibility of a technology platform for the consistent and large-scale production of cell-free reagents from multiple organisms, including methodologies for characterization and validation. Develop detailed analysis of the cell-free platform’s predicted performance characteristics including, but not limited to, total volume of reagent to be produced, batch volume and variability, organisms to be utilized, cost per unit, and distribution format. Include analysis of predicted performance relative to current standard practices. Define key component technological milestones and metrics and establish the minimum performance goals necessary to achieve successful execution of the cell-free platform. Phase I deliverables will include: a detailed analysis of the proposed platform, a technical report detailing experiments and results supporting the feasibility of the approach, and defined milestones and metrics as appropriate for the program goals. Also included with the Phase I deliverables is a Phase II plan for transitioning initial designs and proof-of-concept experiments into protocols that are sufficiently robust and reproducible to be viable as commercial technologies. PHASE II: Finalize the design from Phase I and initiate the development and production of the cell-free platform. Establish appropriate performance parameters through experimentation to determine the efficaciousness, robustness, and fidelity of the approach being pursued. Develop, demonstrate, and validate the reagents and protocols necessary to meet the key metrics as defined for the program, and provide an experimentally validated comparison of the new methods relative to competing state-of-the-art processes. Phase II deliverables include a prototype set of cell-free reagents, including for new organismal systems, and valid test data, appropriate for a commercial production path. PHASE III: The widespread availability and use of cell-free systems will further enable the rapid engineering and optimization of biologically-based manufacturing platforms for the production of previously inaccessible technologies and products, and will facilitate the rapid prototyping of multi-pathway metabolic designs necessary for the engineering of complex biological systems. This will enable DoD to leverage the unique and powerful attributes of biology to solve challenges associated with production of new materials, novel capabilities, fuels, and medicines, while providing novel solutions and enhancements to military needs and capabilities. The successful development of reliable and distributable cell-free platforms for rapidly prototyping biological systems will have widespread applications across the biotechnology and pharmaceutical industries including rapid, optimized production of high value chemicals, industrial enzymes, diagnostics, and therapeutics. These cell-free platforms will be impactful for industrial biotechnology and pharmaceutical firms, as well as government and academic research-scale operations.
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