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Nature BiotechnologySynthetic BiologyPeer-reviewed journal article

Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription

Jaeyoung Kirsten JungiD, Khalid K. AlamiD, Matthew S. VerosloffiD, Daiana A. CapdevilaiD, Morgane DesmauiD, Phillip R. ClaueriD, Jeong Wook LeeiD, Peter Quoc NguyeniD, Pablo PasténiD, Sandrine J. MatiasekiD, Jean‐François GaillardiD, David P. GiedrociD, James J. CollinsiD, Julius B. LucksiD

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Abstract

Synthetic biology has enabled the development of powerful nucleic acid diagnostic technologies for detecting pathogens and human health biomarkers. Here we expand the reach of synthetic biology-enabled diagnostics by developing a cell-free biosensing platform that uses RNA output sensors activated by ligand induction (ROSALIND) to detect harmful contaminants in aqueous samples. ROSALIND consists of three programmable components: highly-processive RNA polymerases, allosteric transcription factors, and synthetic DNA transcription templates. Together, these components allosterically regulate the in vitro transcription of a fluorescence-activating RNA aptamer: in the absence of a target compound, transcription is blocked, while in its presence a fluorescent signal is produced. We demonstrate that ROSALIND can be configured to detect a range of water contaminants, including antibiotics, toxic small molecules, and metals. Our cell-free biosensing platform, which can be freeze-dried for field deployment, creates a new capability for point-of-use monitoring of molecular species to address growing global crises in water quality and human health.

In-depth summary

Claude Sonnet 4.6 · max effort · 8 Nov 2026 · from the full text of version 2 (2 May 2019) · CC BY 4.0

A cell-free platform pairing T7 RNA polymerase, allosteric transcription factors and DNA templates yields fluorescent RNA output to detect antibiotics, small molecules and metals in water.

Chemical water contaminants are usually measured with costly, centralized instrumentation, and existing synthetic biology diagnostics mostly target nucleic acids. This work offers a defined, translation-free, freeze-dryable transcription sensor format that needs no living cells.

Key results

  1. ShownAn RNA aptamer output made by in vitro transcription appears faster and brighter than a protein reporter, and the homemade reaction is cheap.

    3WJdB fluorescence rose immediately, whereas sfGFP (PURE system) needed at least nine minutes before detectable signal. Over four hours 3WJdB gave more fluorescence. In-house IVT cost <1 USD per reaction, a fifth of a commercial IVT kit and a twenty-fifth of the PURE kit. · In vitro transcription of a…

  2. ShownA TetR operator placed a short distance downstream of the T7 promoter gives tight, ligand-reversible control of transcription.

    A 4 BP spacer gave >100-fold repression over four hours without background subtraction. A 25-fold excess of TetR dimer (625 nM) over 25 nM template was the minimum for tight regulation. Half-maximal induction by aTc fell between 1 and 2.5 µM. · In vitro transcriptions can be configured into…

  3. ShownSwapping the transcription factor and operator reconfigures the platform for many ligand classes, and RNA kleptamer circuitry converts aporepressors into activating sensors.

    12 aTFs were used to sense 17 ligands. Detected levels: Zn(II) 10 µM (0.65 ppm, SmtB), Cu 10 µM (0.6 ppm, CsoR), Cd 10 µM (1.12 ppm) and Pb 51.8 µM (10.7 ppm) (CadC). AdcR plus 30 µM Zn(II) activated signal via KB2 inversion. Four representative ligand-aTF pairs showed minimal cross-talk. · RNA circuitry can invert transcription factor…; ROSALIND can be modularly reconfigured to sense…

  4. ShownSensitivity can be moved toward regulatory limits by adjusting aTF and DNA template amounts: more CsoR desensitizes the copper sensor, and less CadC sensitizes the lead sensor.

    Raising CsoR tetramer from 2.5 µM to 7.5 µM (25 nM DNA) shifted copper activation near the 1.3 ppm threshold. Lead activation near 250 ppb at 1.5 µM CadC dimer (guideline 15 ppb) moved to tens of ppb at 0.5 µM CadC, which used 10 nM DNA template. · ROSALIND can be tuned and applied to real-world…; Figure legends

  5. SuggestedFreeze-dried copper and lead sensors worked after rehydration with real water matrices, including two copper-rich Chilean streams, with a handheld illuminator for visual readout.

    Lab, fountain and Lake Michigan water spiked with 2 ppm Pb or 1.3 ppm Cu gave detectable signal within four hours, with lower signal than lab water. Chilean samples (6.9-8.5 ppm Cu by FAAS) gave clear signal at 1:8 dilution (0.88 and 1.05 ppm). · ROSALIND can be tuned and applied to real-world…; DISCUSSION

Methods, data and reproducibility

Allosteric transcription factors gate T7 RNAP transcription of a fluorescence-activating Broccoli aptamer from operator-bearing DNA templates, read on a plate reader or by eye. RNA kleptamers invert aporepressor responses; reactions are freeze-dried with sugars.

  • ROSALIND in vitro transcription reactions with purified aTFs and ligands, plate reader fluorescence converted to FITC equivalents · n = 3 experimental replicates per condition · Addgene plasmid #87308 (pUC19-T7-3WJdB-T backbone)
  • FITC NIST-traceable standard dilution series for fluorescence calibration · n = 12 samples X 9 replicates = 108 samples
  • Laboratory-grade, fountain drinking water and Lake Michigan water spiked with copper or lead · n = 3 water sources, 3 replicates
  • Filtered stream water from Yerba Loca Creek and Toro River, Chile, serially diluted; copper quantified by FAAS · n = 2 streams, 200 mL each; each dilution measured three times
Methods & tools
T7 RNA polymerase, 3WJdB Broccoli aptamer with DFHBI-1T, NEB HiScribe T7 Quick High Yield RNA Synthesis Kit, PURE cell-free system (sfGFP reporter), KB2 RNA kleptamer, Synergy H1 plate reader (BioTek), Flame atomic absorption spectroscopy (PerkinElmer PinAAcle 500), 3D-printed handheld LED illuminator, Gibson Assembly, FPLC (AKTAxpress) with Ni-NTA and size exclusion
Reagents & environment
E. coli Rosetta 2(DE3)pLysS (Novagen #71401) for protein expression; NEB Turbo (#C2984) for cloning; pET-28c vectors; Addgene #87308 backbone; DFHBI-1T (Tocris #5610); NEB HiScribe kit #E2050S.
Experimental conditions
Synergy H1 plate reader, 384-well black plate, 3 min intervals, excitation 472 nm and emission 507 nm at 37° C; overnight lyophilization at 0.04 millibar with a -85° C condenser.
Reproducibility gaps
Plasmid sequences, protocols and device files are in supplementary data files, with no repository URLs printed in the text. Provisional patent applications are declared.
Limitations (5)
  • The platform covers aTFs that act by simple ligand-mediated release from DNA. Factors that recruit additional transcription components (e.g. sigma-54) or alter DNA topology are not supported. · stated by the authors
  • Signals were lower in drinking, lake and Chilean water than in laboratory water, possibly due to transcription inhibitors or fluorescence quenchers. Undiluted Chilean samples gave weak signal, so serial dilution was needed. · stated by the authors
  • Many aTFs sense several related ligands, so a sensor reports a compound family or element class rather than a single analyte. · stated by the authors
  • Specificity was checked for only four representative aTF-ligand pairs, and no formal detection limits or blinded validation are reported. Real-sample testing covered spiked waters and two Chilean streams. · noted in this summary
  • Reported sensitivities for salicylate and benzalkonium chloride lie in the high micromolar to millimolar range, and quantitative readout depends on a plate reader rather than the visual device alone. · noted in this summary

Read this if you want design rules for building cell-free, transcription-only chemical biosensors that can be freeze-dried and tuned to regulatory thresholds.

Citations
405
FWCI
16.29
Field percentile
top 1%

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