Overview¶
The Base Cell is Base Cytosol encapsulated in Base Membrane via Encapsulation: Phase Transfer. The Base Cell is deployed in a glucose outer solution and represents the default synthetic cell for the Nucleus distribution. Base Cells have red fluorescent membranes (Liss-Rhod PE). With pOpen-deGFP as their template DNA, they express GFP over time if functioning correctly.
Overview of Base Cell, composed of Base Cytosol encapsulated with Base Membrane. Constitutive components of each module schematically depicted.
Reference Composition¶
The inner solution encapsulated into the Base Cell is Base Cytosol at reaction concentration, with Optiprep for phase transfer. Template DNA is optional; the cell was validated with pOpen-deGFP DNA as a reporter.
| Component | Input concentration | Final concentration | Volume for one reaction (µL) |
|---|---|---|---|
| SMix | 3.33x | 1x | 12 |
| PMix | 15 mg/mL | 1.80 mg/mL | 4.8 |
| Ribosomes | 10 µM | 1.8 µM | 7.2 |
| tRNA | 35 mg/mL | 3.5 mg/ml | 4 |
| RNase Inhibitor | 40 000 U/mL | 2000 U/mL | 2 |
| Optiprep | 1.32 mg/µL | 0.043 mg/µL | 1.33 |
Template DNA (optional), e.g. pOpen-deGFP | 124 nM | 3 nM | X, e.g. 0.95 |
| Water | 8.67 − X | ||
| Total volume (µL) | 40 |
| Component | Target (mmol/mol) | Molecular Weight (g/mol) | Stock concentration (mg/mL) | Volume to add (µL) |
|---|---|---|---|---|
| POPC | 699.3 | 760.076 | 25 | 162.01 |
| Cholesterol | 299.7 | 386.654 | 50 | 17.66 |
| Liss-Rhod PE | 1 | 1301.71 | 1 | 9.92 |
See Base Membrane for the full membrane spec.
| Component | Concentration |
|---|---|
| Glucose | 1140 mM, a starting recipe: adjust it until its osmotic concentration is 100 mOsm/kg to 150 mOsm/kg below the inner solution’s |
Expected Behavior¶
With pOpen-deGFP as their template DNA, Base Cells express deGFP over time, starting dark and increasing in green fluorescence as translation proceeds. Kinetics data are reported in DevNote bnext-devnotes-base-cell-01.
Combined green (488 nm) and red (561 nm) fluorescence channels. First time point (labeled “t = 0”) corresponds to 30 min after preparation of the inner solution, reflecting the time required to prepare liposomes.
Process¶
Build a Base Cell gives every step at the bench, in order, as one protocol. Base Cells are assembled and encapsulated using Encapsulation: Phase Transfer.
Constituent Modules¶
Base Cytosol — inner solution, encapsulated at reaction concentration
Base Membrane — 70:30 POPC:cholesterol bilayer
Credits¶
Developed by Surendra Yadav (b.next).