Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

One of the foundations of the scientific method is to be able to reproduce experiments and corroborate the results of research that has been done before. However, with the increasing complexities of new technologies and techniques, coupled with the specialisation of experiments, reproducing research findings has become a growing challenge. Clearly, scientific methods must be conveyed succinctly, and with clarity and rigour, in order for research to be reproducible. Here, we propose steps to help increase the transparency of the scientific method and the reproducibility of research results: specifically, we introduce a peer-review oath and accompanying manifesto. These have been designed to offer guidelines to enable reviewers (with the minimum friction or bias) to follow and apply open science principles, and support the ideas of transparency, reproducibility and ultimately greater societal impact. Introducing the oath and manifesto at the stage of peer review will help to check that the research being published includes everything that other researchers would need to successfully repeat the work. Peer review is the lynchpin of the publishing system: encouraging the community to consciously (and conscientiously) uphold these principles should help to improve published papers, increase confidence in the reproducibility of the work and, ultimately, provide strategic benefits to authors and their institutions.

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.