Copper sensor (ACE1 + CRISPRa)
Detects copper in water using the native yeast copper-responsive activator ACE1 and its CUP1 promoter, gating a CRISPRa circuit driving a fluorescent reporter, in GRAS baker's yeast.
Environmental contaminantBSL-1 chassisGRAStemplatecopperheavy-metalwaterenvironmentaleukaryoticyeastCRISPRa
Input
Copper Cu(II)/Cu(I)
Environmental contaminant
→
Sense
CRISPRa-activation
dCas9-VPR (eukaryotic CRISPRa activator)
→
Chassis
Saccharomyces cerevisiae (BY4741)
BSL-1
→
Output
mCherry
fluorescent
What it detects
- Analyte
- Copper Cu(II)/Cu(I) — ACE1/CUP1 responds across µM copper
- Category
- Environmental contaminant
- Signal
- Bioavailable copper in water
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-VPR (eukaryotic CRISPRa activator)
- Analyte sensor
- Copper-bound ACE1 activates the CUP1 promoter (PCUP1).
Signal flow
Cu -> ACE1 activates PCUP1 -> sgRNA -> dCas9-VPR amplifies the reporter (CRISPRa) -> fluorescence rises with copper.Safe chassis
Saccharomyces cerevisiae (BY4741) — Saccharomyces cerevisiae
GRAS baker's yeast — the model eukaryote. Being a eukaryote, it can host human nuclear receptors and GPCRs, unlocking sensing of steroids, hormones, and other eukaryotic-specific ligands that bacteria cannot detect. dCas9 CRISPRi/CRISPRa are well established.
BSL-1GRAS · FDA GRAS
Genetic parts
| Part | Role | Source / id |
|---|---|---|
| ACE1 regulator | regulator | Native S. cerevisiae ACE1 (copper-responsive activator) |
| PCUP1 promoter | promoter | S. cerevisiae CUP1 promoter |
| sgRNA | sgRNA | designed against / upstream of the reporter promoter |
| sgRNA scaffold (SpCas9) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC | sgRNA | Standard SpCas9 scaffold |
| dCas9-VPR | dCas9 | Yeast CRISPRa (dCas9-VPR) |
| mCherry | reporter | standard fluorophore |
Output & readout
- Type
- fluorescent
- Reporter
- mCherry
- Readout
- Red fluorescence (plate reader)
- Positive result
- Fluorescence increases with copper.
Performance
- Device validated
- No — design template (parts validated individually)
ACE1/CUP1 is a classic copper-responsive yeast system; integrated CRISPRa device is a design template.
Safety
- Biosafety level
- BSL-1 (non-pathogenic chassis)
- GRAS chassis
- Yes
- Biocontainment
- Auxotrophic markers; GRAS yeast host.
- Field-deployable
- Yes (with containment)
GRAS baker's yeast.
Build & run
| # | Stage | Step |
|---|---|---|
| 1 | design | Design sgRNA Target/position the sgRNA; check S. cerevisiae off-targets. |
| 2 | assembly | Assemble units Sensor -> sgRNA; dCas9; reporter. Use the host's standard toolkit. |
| 3 | transformation | Transform S. cerevisiae Transform, select, and confirm low background. |
| 4 | induction | Expose to sample Incubate with the sample plus a copper standard curve. |
| 5 | readout | Read result Relate signal to concentration. |
Source & parts
- Design
- Design template combining the native yeast ACE1/CUP1 copper sensor with CRISPRa
- Parts validated in
- Thiele / ACE1-CUP1 copper regulation (yeast)
- Yeast dCas9-VPR CRISPRa
- License
- Parts per their original sources; design template CC BY 4.0