Tetracycline sensor (TetR + CRISPRi)
Detects tetracycline antibiotic contamination in water and food. The classic TetR sensor gates a CRISPRi circuit driving a visible pigment, in safe E. coli K-12.
Chemical / metaboliteBSL-1 chassistemplatetetracyclineantibioticantibiotic-pollutionwaterfood-safetychemicalenvironmentalCRISPRi
Input
Tetracycline / anhydrotetracycline (aTc)
Chemical / metabolite
→
Sense
CRISPRi-repression
dCas9 (S. pyogenes, catalytically dead)
→
Chassis
E. coli K-12 (MG1655)
BSL-1
→
Output
amilCP
pigment
What it detects
- Analyte
- Tetracycline / anhydrotetracycline (aTc) — TetR responds from low nM aTc
- Category
- Chemical / metabolite
- Signal
- Tetracycline-class antibiotic residues in water, food, and wastewater
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
- CRISPRi-repression · NOT logic
- Cas protein
- dCas9 (S. pyogenes, catalytically dead)
- Analyte sensor
- TetR de-represses the Ptet promoter on binding tetracycline/aTc.
Signal flow
Tetracycline -> TetR releases Ptet -> transcribes an anti-pigment sgRNA -> CRISPRi represses a constitutive amilCP cassette -> pigment fades with antibiotic (NOT). Pair an inverter for colour-on.Safe chassis
E. coli K-12 (MG1655) — Escherichia coli
The non-pathogenic laboratory workhorse. K-12 strains have lost the ability to colonize the human gut and are the reference BSL-1 host for genetic engineering, with the deepest tooling of any bacterial chassis.
BSL-1
Genetic parts
| Part | Role | Source / id |
|---|---|---|
| TetR regulator Tetracycline/aTc-responsive repressor; one of the best-characterised regulators. | regulator | Tn10 tet operon (also a Marionette/standard sensor) |
| Ptet promoter De-repressed by tetracycline-bound TetR. | promoter | BBa_R0040 |
| Anti-pigment sgRNA | sgRNA | designed against the amilCP promoter |
| sgRNA scaffold (SpCas9) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC | sgRNA | Standard SpCas9 scaffold |
| dCas9 | dCas9 | Qi et al. 2013 (CRISPRi) |
| amilCP chromoprotein | reporter | BBa_K592009 |
Output & readout
- Type
- pigment
- Reporter
- amilCP
- Readout
- Visible pigment (naked eye / smartphone)
- Positive result
- Pigment change reports tetracycline contamination.
Performance
- Limit of detection
- TetR module: low-nM aTc (module-validated).
- Dynamic range
- ~1-1000 nM tetracycline/aTc
- Response time
- ~150 min
- Device validated
- No — design template (parts validated individually)
TetR/Ptet is among the most characterised regulator-promoter pairs in biology; integrated CRISPRi device is a design template.
Safety
- Biosafety level
- BSL-1 (non-pathogenic chassis)
- GRAS chassis
- No
- Biocontainment
- Lab/contained; add kill-switch for field use.
- Field-deployable
- Yes (with containment)
E. coli K-12 non-pathogenic BSL-1 host; relevant to antibiotic-pollution surveillance. Note: do not select the host with tetracycline resistance.
Build & run
| # | Stage | Step |
|---|---|---|
| 1 | design | Design anti-pigment sgRNA Target the amilCP promoter; check host off-targets. |
| 2 | assembly | Assemble units TU1: TetR + Ptet -> sgRNA. TU2: dCas9 + constitutive amilCP. Avoid TetR-resistance markers. |
| 3 | transformation | Transform E. coli K-12 Select (non-tet marker); confirm baseline pigment without antibiotic. |
| 4 | induction | Expose to sample Add water/food sample + tetracycline standard curve. |
| 5 | readout | Score colour Compare pigment to the curve. |
Source & parts
- Design
- Design template combining the classic TetR tetracycline sensor with E. coli CRISPRi
- Parts validated in
- Hillen & Berens / TetR tetracycline regulation (Tn10)
- Qi et al. 2013, Cell (CRISPRi)
- License
- Parts per their original sources; design template CC BY 4.0