Cadmium sensor (CadR + CRISPRi)
Detects cadmium in industrial wastewater. A CadR/CadC metal-responsive regulator gates a CRISPRi circuit driving a fluorescent reporter, in the robust GRAS industrial chassis Corynebacterium glutamicum.
Environmental contaminantBSL-1 chassisGRAStemplatecadmiumheavy-metalindustrialwastewaterenvironmentalCRISPRi
Input
Cadmium Cd(II)
Environmental contaminant
→
Sense
CRISPRi-repression
dCas9 (S. pyogenes, catalytically dead)
→
Chassis
Corynebacterium glutamicum (ATCC 13032)
BSL-1
→
Output
sfGFP
fluorescent
What it detects
- Analyte
- Cadmium Cd(II) — CadR/CadC respond from sub-µM to µM Cd(II)
- Category
- Environmental contaminant
- Signal
- Cadmium in industrial 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
- A CadR/CadC-family metalloregulator switches the cad promoter on cadmium binding.
Signal flow
Cd(II) -> CadR switches Pcad -> anti-reporter sgRNA -> CRISPRi inverts a constitutive reporter (NOT). Pair an inverter for turn-on.Safe chassis
Corynebacterium glutamicum (ATCC 13032) — Corynebacterium glutamicum
A GRAS industrial workhorse used for food-grade amino-acid production at megaton scale. Robust, stress-tolerant, and easy to grow at high density — well suited to industrial-process and chemical monitoring.
BSL-1GRAS · EFSA QPS
Genetic parts
| Part | Role | Source / id |
|---|---|---|
| CadR regulator | regulator | Cadmium-resistance (cad) operon regulator |
| Pcad promoter | promoter | cad operon promoter |
| sgRNA | sgRNA | designed against / upstream of the reporter promoter |
| sgRNA scaffold (SpCas9) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC | sgRNA | Standard SpCas9 scaffold |
| dCas9 | dCas9 | Cleto et al. 2016 (CRISPRi in C. glutamicum) |
| sfGFP | reporter | Pedelacq et al. 2006 |
Output & readout
- Type
- fluorescent
- Reporter
- sfGFP
- Readout
- Green fluorescence (plate reader)
- Positive result
- Fluorescence change reports cadmium.
Performance
- Device validated
- No — design template (parts validated individually)
CadR/CadC cadmium sensing and C. glutamicum CRISPRi are validated separately; integrated device is a design template. C. glutamicum tolerates harsh industrial samples.
Safety
- Biosafety level
- BSL-1 (non-pathogenic chassis)
- GRAS chassis
- Yes
- Biocontainment
- Auxotrophic containment; GRAS industrial host.
- Field-deployable
- Yes (with containment)
GRAS, robust for industrial wastewater monitoring.
Build & run
| # | Stage | Step |
|---|---|---|
| 1 | design | Design sgRNA Target/position the sgRNA; check C. glutamicum off-targets. |
| 2 | assembly | Assemble units Sensor -> sgRNA; dCas9; reporter. Use the host's standard toolkit. |
| 3 | transformation | Transform C. glutamicum Transform, select, and confirm low background. |
| 4 | induction | Expose to sample Incubate with the sample plus a cadmium standard curve. |
| 5 | readout | Read result Relate signal to concentration. |
Source & parts
- Design
- Design template combining a CadR cadmium-sensing module with C. glutamicum CRISPRi
- Parts validated in
- CadR/CadC cadmium-resistance literature
- Cleto et al. 2016 (CRISPRi in C. glutamicum)
- License
- Parts per their original sources; design template CC BY 4.0