Gut sulfide sensor (CstR + CRISPRa)
An ingestible sensor for hydrogen sulfide / persulfide, a gut-microbial metabolite linked to colitis and colorectal risk. The CstR persulfide-sensing repressor gates a CRISPRa circuit driving a fluorescent reporter, in the probiotic E. coli Nissle 1917.
Clinical / gut biomarkerBSL-1 chassistemplateclinical-guthydrogen-sulfidepersulfidemetaboliteprobioticNissleCRISPRa
Input
Hydrogen sulfide / persulfide (H2S)
Clinical / gut biomarker
→
Sense
CRISPRa-activation
dCas9-ω (CRISPRa activator)
→
Chassis
E. coli Nissle 1917
BSL-1
→
Output
sfGFP
fluorescent
What it detects
- Analyte
- Hydrogen sulfide / persulfide (H2S) — CstR responds to µM persulfide
- Category
- Clinical / gut biomarker
- Signal
- Luminal hydrogen sulfide / reactive sulfur species from gut microbial metabolism
Genetic circuit
⤢ click to enlarge
Genetic construct (SBOL)
The DNA construct as transcription units, drawn with SBOL Visual part glyphs.
⤢ click to enlarge
CRISPR sensing mechanism
- Strategy
- CRISPRa-activation · amplifier logic
- Cas protein
- dCas9-ω (CRISPRa activator)
- Analyte sensor
- CstR is a persulfide-sensing repressor; reactive sulfur species crosslink its cysteines and release it from its operator.
Signal flow
H2S/persulfide -> CstR releases Pcst -> transcribes an sgRNA -> dCas9-activator amplifies a fluorescent reporter (CRISPRa) -> fluorescence rises with gut sulfide.Safe chassis
E. coli Nissle 1917 — Escherichia coli
A probiotic E. coli used in humans for over a century (Mutaflor). Colonizes the gut safely, making it the chassis of choice for clinical / gut biomarker biosensors.
BSL-1probiotic
Genetic parts
| Part | Role | Source / id |
|---|---|---|
| CstR regulator Reactive-sulfur-species-responsive repressor; requires porting into E. coli Nissle. | regulator | Staphylococcus/Bacillus cst operon (persulfide sensor) |
| Pcst promoter De-repressed by persulfide-modified CstR. | promoter | cst operon operator/promoter |
| Reporter-activating sgRNA | sgRNA | designed for CRISPRa upstream of a weak reporter promoter |
| sgRNA scaffold (SpCas9) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC | sgRNA | Standard SpCas9 scaffold |
| dCas9-ω activator | dCas9 | Bikard et al. 2013 (CRISPRa) |
| sfGFP reporter Recoverable from stool for non-invasive readout. | reporter | Pedelacq et al. 2006 |
Output & readout
- Type
- fluorescent
- Reporter
- sfGFP
- Readout
- Fluorescence (flow cytometry on recovered cells)
- Positive result
- Fluorescent-cell fraction rises with luminal sulfide.
Performance
- Limit of detection
- CstR module: µM persulfide (module-validated).
- Dynamic range
- Physiological gut sulfide range
- Response time
- ~180 min
- Device validated
- No — design template (parts validated individually)
CstR persulfide sensing and CRISPRa are validated separately; integrated device is a design template requiring CstR porting to Nissle.
Safety
- Biosafety level
- BSL-1 (non-pathogenic chassis)
- GRAS chassis
- No
- Biocontainment
- Probiotic E. coli Nissle host; add thyA/dapA auxotrophy for gut-restricted containment.
- Field-deployable
- Lab / supervised use
Probiotic chassis with a human-safety record; research / supervised clinical use only.
Build & run
| # | Stage | Step |
|---|---|---|
| 1 | design | Port CstR + design sgRNA Reconstitute CstR/Pcst in Nissle; design the CRISPRa sgRNA. |
| 2 | assembly | Assemble units TU1: CstR. TU2: Pcst -> sgRNA. TU3: dCas9-omega. TU4: weak promoter -> sfGFP. Low-copy vector. |
| 3 | transformation | Transform E. coli Nissle 1917 Select; add auxotrophic containment. |
| 4 | induction | Validate in vitro Confirm sulfide-donor response before any animal work. |
| 5 | readout | Recover and measure Recover cells from stool; quantify fluorescence. |
Source & parts
- Design
- Design template combining the CstR persulfide sensor with a dCas9 CRISPRa circuit in E. coli Nissle
- Parts validated in
- Luebke / Giedroc 2014 / CstR persulfide sensing
- Bikard et al. 2013, NAR (CRISPRa)
- License
- Parts per their original sources; design template CC BY 4.0