Abstract
Industrialization and failing infrastructure have led to a growing number of irreversible health conditions resulting from chronic lead exposure. While state-of-the-art analytical chemistry methods provide accurate and sensitive detection of lead, they are too slow, expensive, and centralized to be accessible to many. Cell-free biosensors based on allosteric transcription factors (aTFs) can address the need for accessible, on-demand lead detection at the point of use. However, known aTFs, such as PbrR, are unable to detect lead at concentrations regulated by the Environmental Protection Agency (24-72 nM). Here, we develop a rapid cell-free platform for engineering aTF biosensors with improved sensitivity, selectivity, and dynamic range characteristics. We apply this platform to engineer PbrR mutants for a shift in limit of detection from 10 μM to 50 nM lead and demonstrate use of PbrR as a cell-free biosensor. We envision that our workflow could be applied to engineer any aTF.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3003-3012 |
| Number of pages | 10 |
| Journal | ACS synthetic biology |
| Volume | 13 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 20 2024 |
Funding
This work was supported by the Army Research Laboratory and the Army Research Office (W911NF-23-1-0334 and W911NF-22-2-0246), AFOSR (FA9550-23-1-0420), and National Science Foundation (2319427 and 2310382 to J.B.L.). J.B.L. was supported by a John Simon Guggenheim Fellowship. H.M.E. was supported by a National Defense Science and Engineering Graduate Research Fellowship (F-6669029987) and Paul and Daisy Soros Fellowship for New Americans. B.M.W. was supported by a National Science Graduate Research Fellowship (DGE-2234667).
Keywords
- cell-free gene expression
- detection
- diagnostic
- lead
- protein engineering
- synthetic biology
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)