Alkane/oil sensor (AlkS + CRISPRa)
Detects medium-chain alkanes (diesel / crude-oil contamination). The AlkS regulator gates a CRISPRa circuit driving a fluorescent reporter, in the solvent-tolerant chassis P. putida KT2440.
Environmental contaminantBSL-1 chassistemplatealkaneoildieselhydrocarbonpollutantsoilwaterenvironmentalCRISPRa
Input
Medium-chain alkanes (e.g. octane)
Environmental contaminant
→
Sense
CRISPRa-activation
dCas9-ω (CRISPRa activator)
→
Chassis
Pseudomonas putida KT2440
BSL-1
→
Output
sfGFP
fluorescent
What it detects
- Analyte
- Medium-chain alkanes (e.g. octane) — AlkS/PalkB responds across µM alkane
- Category
- Environmental contaminant
- Signal
- Alkane/oil contamination in soil and water (spill monitoring)
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
- AlkS binds alkanes and activates the PalkB promoter of the alkane-degradation operon.
Signal flow
Alkane -> AlkS activates PalkB -> transcribes an sgRNA -> dCas9-activator amplifies a fluorescent reporter (CRISPRa) -> fluorescence rises with hydrocarbon contamination.Safe chassis
Pseudomonas putida KT2440 — Pseudomonas putida
A certified safety-strain soil bacterium with exceptional solvent/stress tolerance and diverse metabolism. The premier chassis for environmental biosensing of pollutants and heavy metals in soil and water.
BSL-1
Genetic parts
| Part | Role | Source / id |
|---|---|---|
| AlkS regulator Alkane-responsive activator. | regulator | Pseudomonas putida/oleovorans OCT plasmid alk operon |
| PalkB promoter Activated by alkane-bound AlkS. | promoter | alk operon 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 | Pedelacq et al. 2006 |
Output & readout
- Type
- fluorescent
- Reporter
- sfGFP
- Readout
- Green fluorescence (field fluorimeter)
- Positive result
- Fluorescence increases with alkane/oil contamination.
Performance
- Limit of detection
- AlkS/PalkB module: µM alkane (module-validated).
- Dynamic range
- ~1-100 µM alkane
- Response time
- ~180 min
- Device validated
- No — design template (parts validated individually)
AlkS is a classic alkane sensor and PalkB a widely used inducible promoter; integrated CRISPRa device is a design template. P. putida natively tolerates solvents.
Safety
- Biosafety level
- BSL-1 (non-pathogenic chassis)
- GRAS chassis
- No
- Biocontainment
- P. putida KT2440 certified HV1 safety host; add containment for field use.
- Field-deployable
- Yes (with containment)
Solvent-tolerant chassis suited to spill sites.
Build & run
| # | Stage | Step |
|---|---|---|
| 1 | design | Design CRISPRa sgRNA Target a weak reporter promoter; check host off-targets. |
| 2 | assembly | Assemble units TU1: AlkS + PalkB -> sgRNA. TU2: dCas9-omega. TU3: weak promoter -> sfGFP. SEVA vectors. |
| 3 | transformation | Transform P. putida KT2440 Electroporate; select; confirm alkane-dependent activation. |
| 4 | induction | Expose to sample Add soil eluate/water + alkane standard curve. |
| 5 | readout | Measure fluorescence Interpolate alkane from the curve. |
Source & parts
- Design
- Design template combining the AlkS alkane-sensing module with P. putida CRISPRa
- Parts validated in
- de Lorenzo et al. / AlkS-PalkB alkane regulation (Pseudomonas)
- Bikard et al. 2013, NAR (CRISPRa)
- License
- Parts per their original sources; design template CC BY 4.0