Overview¶
The pH Sensing Cell is a synthetic cell that expresses phospholipase A1 (PLA1) when its outer solution is acidic, at about pH 6.0 to 6.5. The pH-Sensing Detector gates the expression: acidification releases a trigger strand, and the free trigger strand opens the toehold switch in front of the PLA1 gene. PLA1 disrupts phospholipid membranes: the membrane of the cell that expresses it, and the membranes of neighboring liposomes, which then release their contents. Cy5 in the inner solution marks the cell. A cell with an intact membrane is bright in the Cy5 channel.
The cell is Base Cytosol with the pH-Sensing Detector added, encapsulated in a POPC/cholesterol membrane by Protocol: Inverted Emulsion. The membrane has its own ratio of lipids, given in the Membrane tab, and it differs from the ratio of the Base Membrane: POPC/Chol.
Reference Composition¶
DNA of the pH Sensing Cell.
| Name | Length | Supply | File |
|---|---|---|---|
pT7-toehold9-PLA1-linear | 1203 bp | Linear DNA template, added to the cytosol at 2 nM | pT7 |
pH-responsive-ssDNA-2 | 49 nt | Single-stranded oligo, annealed with the trigger strand | pH |
trigger-ssDNA-3 | 36 nt | Single-stranded oligo, annealed with the pH-responsive strand | trigger-ssDNA-3.gb |
The inner solution is Base Cytosol at reaction concentration, with the PLA1 template, the annealed duplex, Optiprep, RNase inhibitor and Cy5 added.
Inner solution of the pH Sensing Cell.
| Component | Input concentration | Final concentration | Volume for one preparation (µL) |
|---|---|---|---|
| SMix | 3.33× | 1× | 6 |
| PMix | 15 mg/mL | 1.80 mg/mL | 2.4 |
| Ribosomes | 10 µM | 1.8 µM | 3.6 |
| tRNA | 35 mg/mL | 3.5 mg/mL | 2 |
pT7-toehold9-PLA1-linear DNA template | 80 nM | 2 nM | 0.5 |
| pH-responsive : trigger ssDNA duplex (3:1), annealed | 25 µM, as the trigger strand | 4.625 µM | 3.7 |
| Optiprep | 1000 mL/L | 45 mL/L | 0.9 |
| RNase inhibitor | 40 000 U/mL | 1000 U/mL | 0.5 |
| Cy5 (sulfo-Cyanine5 carboxylic acid) | 100 µM | 2 µM | 0.4 |
| Total volume (µL) | 20 |
The duplex is annealed in IDT duplex buffer by Anneal pH Trigger Duplex.
Membrane of the pH Sensing Cell. Volumes are for the lipid stocks that make 2.5 µmol of total lipid.
| Component | Target (mmol/mol) | Molecular Weight (g/mol) | Stock concentration (mg/mL) | Volume to add (µL) |
|---|---|---|---|---|
| POPC | 899.1 | 760.076 | 25 | 68.34 |
| Cholesterol | 99.9 | 386.654 | 50 | 1.93 |
| Liss-Rhod PE | 1 | 1301.71 | 1 | 3.25 |
Suspension buffer, the outer solution in which the pH Sensing Cell is prepared. 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¶
Cells¶
The pH Sensing Cell is expected to keep its Cy5 at pH 7.6 and to lose it at pH 6.3. In solution after 13 h at 37 °C, cells at pH 7.6 kept Cy5, and cells at pH 6.3 lost it, which is consistent with PLA1 expression disrupting the membrane. Data are in the Colorimetric pH Sensor, read by fluorescence microscopy in the Rhodamine and Alexa Fluor 647 channels.
Requirements¶
Requires pT7 transcription and translation (e.g. Base Cytosol), supplied here by the Base Cytosol in the cell.
Implementations¶
Processes¶
Anneal pH Trigger Duplex makes the duplex of the pH-responsive strand and the trigger strand.
Protocol: Inverted Emulsion makes the cells, from 5 mL of lipid-in-oil at 0.5 mM total lipid.
Embedding: Thermal Setting places the cells in a gel.
Materials¶
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| RNase Inhibitor, Murine | Reagent | RNase Inhibitor, Murine | NEB | M0314S | $87.00 | -25 °C to -15 °C | link |
| Cy5 | Reagent | Sulfo-Cyanine5 carboxylic acid | Lumiprobe | 13390 | $99.00 | -20 °C | link |
| HEPES | Salts | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4 °C to 30 °C | link |
| IDT duplex buffer | Reagent | Nuclease Free Duplex Buffer (10 x 2 mL) | IDT | 11-01-03-01 | $17.00 | -20 °C | link |
| Optiprep | Reagent | OptiPrep™ | STEMCELL Technologies | 07820 | $289.00 | RT | 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 |
| Mineral oil, light | Chemical | Mineral oil, BioReagent, for molecular biology, light oil | Millipore Sigma | M5904-500ML | $78.70 | RT | link |
Constituent Modules¶
Base Cytosol — inner solution, encapsulated at reaction concentration
Detector: pH-Sensing — turns acidic pH into PLA1 expression
Credits¶
Developed by Sung-Won Hwang, Samuel Chen and Allen P. Liu (University of Michigan).
Supported by¶
DevNotes that support this specification include:
