"Just add small molecules" cell-free protein synthesis: Combining DNA template and cell extract preparation into a single fermentation.

Cell-free protein synthesis (CFPS) is a versatile biotechnology platform enabling a broad range of applications including clinical diagnostics, large-scale production of officinal therapeutics, small-scale on-demand production of personal magistral therapeutics, and exploratory research. The shelf stability and scalability of CFPS systems also have the potential to overcome cost and infrastructure challenges for distributing and using essential medical tests at home in both high- and low-income countries. However, CFPS systems are often more time-consuming and expensive to prepare than traditional in vivo systems, limiting their broader use. Much work has been done to lower CFPS costs by optimizing cell extract preparation, small molecule reagent recipes, and DNA template preparation. In order to further reduce reagent cost and preparation time, this work presents a CFPS system that does not require separately purified DNA template. Instead, a DNA plasmid encoding the recombinant protein is transformed into the cells used to make the extract, and the extract preparation process is modified to allow enough DNA to withstand homogenization-induced shearing. The finished extract contains sufficient levels of intact DNA plasmid for the CFPS system to operate. For a 10 mL scale CFPS system expressing recombinant sfGFP protein for a biosensor, this new system reduces reagent cost by more than half. This system is applied to a proof-of-concept glutamine sensor compatible with smartphone quantification to demonstrate its viability for further cost reduction and use in low-resource settings.

Engineering At-Home Dilution and Filtration Methods to Enable Paper-Based Colorimetric Biosensing in Human Blood with Cell-Free Protein Synthesis.

Diagnostic blood tests can guide the administration of healthcare to save and improve lives. Most clinical biosensing blood tests require a trained technician and specialized equipment to process samples and interpret results, which greatly limits test accessibility. Colorimetric paper-based diagnostics have an equipment-free readout, but raw blood obscures a colorimetric response which has motivated diverse efforts to develop blood sample processing techniques. This work uses inexpensive readily-available materials to engineer user-friendly dilution and filtration methods for blood sample collection and processing to enable a proof-of-concept colorimetric biosensor that is responsive to glutamine in 50 µL blood drop samples in less than 30 min. Paper-based user-friendly blood sample collection and processing combined with CFPS biosensing technology represents important progress towards the development of at-home biosensors that could be broadly applicable to personalized healthcare.