Javin P. Oza

Cell-free protein synthesis (CFPS) is a powerful platform for rapid protein prototyping and biomanufacturing, but expression of complex proteins remains limited by labor-intensive extract preparation as well as the inability of conventional Escherichia coli extracts to efficiently support disulfide bond formation and prevent protein aggregation. We previously developed a Cell-Free Autoinduction (CFAI) workflow that simplifies extract preparation, however, CFAI-derived extracts retain the folding limitations of standard E. coli systems for producing traditionally difficult proteins. Here, we address these challenges by utilizing extracts enriched with molecular chaperones or Erv1p-based oxidative folding catalysts and combining them through a modular extract mixing strategy to enable independent tuning of the cell-free folding environment. This workflow supplies folding factors through enriched extracts, avoiding recurring purchases of purified supplements and enabling a modeled reagent-cost advantage. To evaluate real-world applications and utility of lysate mixing for protein production, we demonstrate a 10-fold increase in solubility of the aggregation-prone bacterial toxin Colicin M, achieving >95% soluble protein while retaining antimicrobial activity. We further used a Design of Experiments (DoE) framework to optimize combinations of chaperone-enriched and Erv1p-based oxidative folding extracts containing either protein disulfide isomerase (PDI) or DsbC. The optimized compositions produced >95% soluble truncated human tissue plasminogen activator (vtPA), comprising the kringle 2 and protease domains, while preserving enzymatic activity. Together, these results demonstrate that CFAI-derived modular extract mixing provides a tunable, high-throughput strategy with low modeled materials costs for tailoring cell-free biochemical environments for difficult-to-express proteins, expanding CFPS utility for protein engineering and biomanufacturing. Abstract Figure:

Research project planning skills are typically developed through experiential learning rather than systematic instruction. As a result, students struggle with the higher-order work of scoping projects, structuring inquiries, and translating ideas into tractable projects. Experienced researchers face analogous difficulties when conceptualizing new directions or managing execution of complex projects. Limitations aren’t in researcher ability, but the absence of structural frameworks to facilitate the processes involved in project planning and execution. Existing planning frameworks address isolated aspects of research design, yet no single tool integrates problem definition, hypothesis formation, methodological planning, validation criteria, and risk assessment in a format optimized for both initial planning and iterative refinement during execution.Here I present the Research Project Compass to address the structural gaps in how research projects are conceived, refined, communicated, and executed. The Compass is a single-page visual framework that externalizes research planning across seven interconnected spaces: Problem, Claim, Value, Execution, Validation, Strategy & Risk, and Constraints. Drawing on cognitive load theory, design thinking principles, and research on scientific reasoning, the framework reduces cognitive overhead while maintaining visibility of dependencies between project elements. Unlike static planning templates, the Compass functions as a living document that evolves with the research. It supports adaptive hypothesis refinement, maintains focus on core aims during execution, and enables earlier recognition of productive pivots versus unproductive drift. This manuscript presents the theoretical foundations of the Compass framework, explains design principles underlying each space and their logical connections, and provides implementation guidance alongside a visual template. The Compass is designed for use across research disciplines ranging from the sciences, engineering, and the humanities, and is anticipated to help researchers at all career stages.

The fragility of biological systems during storage, transport, and utilization necessitates reliable cold-chain infrastructure and limits the potential of biotechnological applications. In order to unlock the broad applications of existing and emerging biological technologies, we report the development of a novel solid-state storage platform for complex biologics. The resulting solid-state biologics (SSB) platform meets four key requirements: facile rehydration of solid materials, activation of biochemical activity, ability to support complex downstream applications and functionalities, and compatibility for deployment in a variety of reaction formats and environments. As a model system of biochemical complexity, we utilized crudeEscherichia colicell extracts that retain active cellular metabolism and support robust levels of in vitro transcription and translation. We demonstrate broad versatility and utility of SSB through proof-of-concepts for on-demand in vitro biomanufacturing of proteins at a milliliter scale, the activation of downstream CRISPR activity, as well as deployment on paper-based devices. SSBs unlock a breadth of applications in biomanufacturing, discovery, diagnostics, and education in resource-limited environments on Earth and in space.

Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design–build–test–learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field.

Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, allowing for the controlled expression of proteins without reliance on living cells. However, the process of producing the cell extract, a key component of cell-free reactions, can be a bottleneck for new users to adopt CFPS as it requires technical knowledge and significant researcher oversight. Here, we provide a detailed method for implementing a simplified cell extract preparation workflow using CFAI media. We also provide a detailed protocol for the alternative, 2x YPTG media-based preparation process, as it represents a useful benchmark within the cell-free community.

Cell-free systems are a rapidly expanding platform technology with an important role in the engineering of biological systems. The key advantages that drive their broad adoption are increased efficiency, versatility, and low cost compared to in vivo systems. Traditionally, in vivo platforms have been used to synthesize novel and industrially relevant proteins and serve as a testbed for prototyping numerous biotechnologies such as genetic circuits and biosensors. Although in vivo platforms currently have many applications within biotechnology, they are hindered by time-constraining growth cycles, homeostatic considerations, and limited adaptability in production. Conversely, cell-free platforms are not hindered by constraints for supporting life and are therefore highly adaptable to a broad range of production and testing schemes. The advantages of cell-free platforms are being leveraged more commonly by the biotechnology community, and cell-free applications are expected to grow exponentially in the next decade. In this study, new and emerging applications of cell-free platforms, with a specific focus on cell-free protein synthesis (CFPS), will be examined. The current and near-future role of CFPS within metabolic engineering, prototyping, and biomanufacturing will be investigated as well as how the integration of machine learning is beneficial to these applications.

High-school and undergraduate college students may often rely on memorization when learning periodic trends, leading to an ineffective understanding of introductory chemistry concepts like atomic radius, ionization energy, and electron affinity. Comprehension of effective nuclear charge (Zeff) is foundational to a complete understanding of periodic trends. Zeff remains an abstract concept for many students, indicating that a manipulative activity for teaching the phenomenon could be a useful way to explain the concept for greater student comprehension. Here we report a hands-on learning activity to target this knowledge gap using magnetic attraction as an analogy for electrostatic attraction within the atom. This approach enables students to explore the phenomenon of effective nuclear charge at a macroscopic level and apply their learning to periodic trends and related concepts. We anticipate that this activity will fill a long-standing hole in the active learning of chemical principles. Importantly, this activity is low-cost and can be assembled using readily accessible materials to allow implementation in most classrooms and virtual learning environments.

High-school and undergraduate college students may often rely on memorization when learning periodic trends, leading to an ineffective understanding of introductory chemistry concepts like atomic radius, ionization energy, and electron affinity. Comprehension of effective nuclear charge (Zeff) is foundational to a complete understanding of periodic trends. Zeff remains an abstract concept for many students, indicating that a manipulative activity for teaching the phenomenon could be a useful way to explain the concept for greater student comprehension. Here we report a hands-on learning activity to target this knowledge gap using magnetic attraction as an analogy for electrostatic attraction within the atom. This approach enables students to explore the phenomenon of effective nuclear charge at a macroscopic level and apply their learning to periodic trends and related concepts. We anticipate that this activity will fill a long-standing hole in the active learning of chemical principles. Importantly, this activity is low-cost and can be assembled using readily accessible materials to allow implementation in most classrooms and virtual learning environments.