Overview¶
Substrate SUV: CPRG encapsulates the chromogenic substrate chlorophenol red-β-D-galactopyranoside (CPRG) in small unilamellar vesicles (SUVs) with a POPC/cholesterol membrane. It is a substrate population of the pH Cascade. The SUVs are made by Encapsulation: Freeze-Thaw with an extra extrusion through a 400 nm membrane. Intact SUVs keep CPRG inside. A lytic enzyme such as phospholipase A1 (PLA1) disrupts the membrane, and CPRG is released into the outer solution. There, β-galactosidase converts the yellow CPRG into the purple chlorophenol red (CPR).
Reference Composition¶
Inner solution of Substrate SUV: CPRG.
| Component | Final concentration | Volume for one preparation (µL) |
|---|---|---|
| CPRG | not documented | not documented |
| Other components | not documented | not documented |
| Total volume (µL) | 1000 |
Membrane. Volumes are for the lipid stocks that make 2 µmol of total lipid, the same film as Substrate LUV: CPRG.
| Component | Target (mmol/mol) | Molecular Weight (g/mol) | Stock concentration (mg/mL) | Volume to add (µL) |
|---|---|---|---|---|
| POPC | 899.1 | 760.076 | 25 | 54.67 |
| Cholesterol | 99.9 | 386.654 | 50 | 1.55 |
| Liss-Rhod PE | 1 | 1301.71 | 1 | 2.60 |
Suspension buffer, the outer solution after preparation. Its pH is 8, and its osmolarity is 1170 mOsm/kg to 1180 mOsm/kg.
| Component | Concentration |
|---|---|
| Tris1M-HEPES1.15M buffer stock | 466.7 mL/L |
The Tris1M-HEPES1.15M buffer stock is 1 M Tris and 1.15 M HEPES at pH 8, about 2520 mOsm/kg, measured.
Expected Behavior¶
Solution¶
Substrate SUV: CPRG is expected to stay yellow in solution when pH Sensing Cells are present at pH 7.6, 5.5 h after β-galactosidase is added. Data are in the Colorimetric pH Sensor, read by Colorimetric Readout.
Gels¶
Substrate SUV: CPRG is expected to stay yellow in 3.5 mg/mL low-gelling agarose when pH Sensing Cells are present at pH 7.6, 5.5 h after β-galactosidase is added. In 7 mg/mL low-gelling agarose, the color difference between the pH arms and the control was not visible by eye, and the yields of SUVs and cells were lower. Data are in the Colorimetric pH Sensor, read by Colorimetric Readout.
Requirements¶
Requires a membrane-lytic activity to release CPRG (e.g. the pH Sensing Cell, which expresses PLA1).
Requires β-galactosidase in the outer solution to convert released CPRG (e.g. Reporter: LacZ Enzyme).
Implementations¶
Processes¶
Encapsulation: Freeze-Thaw makes the SUVs: the lipid film is hydrated in 1 mL of inner solution, and the suspension is extruded through a 400 nm membrane in 13 passes.
Embedding: Thermal Setting places them in a gel.
Colorimetric Readout develops the color of released CPRG.
Materials¶
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| CPRG | Reagent | Chlorophenol Red-β-D-galactopyranoside | Roche | 10884308001 | $160.00 | -20 °C in water at 20 mg/mL | link |
| Optiprep | Reagent | OptiPrep™ | STEMCELL Technologies | 07820 | $289.00 | RT | link |
| HEPES | Salts | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4 °C to 30 °C | link |
| POPC | Lipid | 16:0-18:1 PC 25 mg/mL | Avanti Lipids | A80557 | $435.00 | -20 °C | link |
| Cholesterol | Lipid | Cholesterol (plant) | Avanti Research | A80100 | $261.00 | -20 °C | link |
| Liss-Rhod PE | Lipid | 18:0 Liss Rhod PE 1 mg/mL | Avanti Lipids | A81179 | $273.47 | -20 °C | link |
| Liposome extruder | Equipment | Mini Extruder Pro Kit with base | Avanti Lipids | A81411 | RT | link | |
| Membrane filter, 400 nm | Consumable | PC Membranes 0.4 µm, 19 mm | Avanti Lipids | A89040 (legacy 610007) | RT | link |
Supported by¶
DevNotes that support this specification include:
