Year: 2024
Type: article
Abstract: The design and optimization of metabolic pathways, genetic systems, and engineered proteins rely on high-throughput assays to streamline design-build-test-learn cycles. However, assay development is a time-consuming and laborious process. Here, we create a generalizable approach for the tailored optimization of automated cell-free gene expression (CFE)-based workflows, which offers distinct advantages over in vivo assays in reaction flexibility, control, and time to data. Centered around designing highly accurate and precise transfers on the Echo Acoustic Liquid Handler, we introduce pilot assays and validation strategies for each stage of protocol development. We then demonstrate the efficacy of our platform by engineering transcription factor-based biosensors. As a model, we rapidly generate and assay libraries of 127 MerR and 134 CadR transcription factor variants in 3682 unique CFE reactions in less than 48 h to improve limit of detection, selectivity, and dynamic range for mercury and cadmium detection. This was achieved by assessing a panel of ligand conditions for sensitivity (to 0.1, 1, 10 μM Hg and 0, 1, 10, 100 μM Cd for MerR and CadR, respectively) and selectivity (against Ag, As, Cd, Co, Cu, Hg, Ni, Pb, and Zn). We anticipate that our Echo-based, cell-free approach can be used to accelerate multiple design workflows in synthetic biology.
Source: ACS Synthetic Biology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: Bill and Melinda Gates Foundation, Paul and Daisy Soros Fellowships for New Americans, Northwestern University, National Defense Science and Engineering Graduate, Chemistry of Life Processes Institute, Air Force Office of Scientific Research, Army Research Office, Army Research Laboratory, National Science Foundation Graduate Research Fellowship Program, Army Contracting Command - Aberdeen Proving Ground
Awards: G1596173563, G2294708377, G299359792, G3161376373, G3352712625, Northwestern University - to empower farmers to monitor their own crops, enable regional quality control of distributed plant material, and create large-scale crop pathogen ..., G3902745504, G4719189355, G5259331294, G5536892680, G585296059, G602718224, G7452299184, G8335976078
OpenAlex ID: W4403191880