An Automated Cell-Free Workflow for Transcription Factor Engineering

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.