| Autor: |
Ekas HM; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Wang B; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Silverman AD; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Lucks JB; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States.; Center for Engineering Sustainability and Resilience, Northwestern University, Evanston, Illinois 60208, United States., Karim AS; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Jewett MC; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States.; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611, United States.; Simpson Querrey Institute, Northwestern University, Chicago, Illinois 60611, United States.; Department of Bioengineering, Stanford University, Stanford, California 94305, United States. |
| Abstrakt: |
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. |
