Here you’ll boot up a simple synthetic cell using commercially available PURE (PURExpress). You’ll need ~$1500 of materials, a microscope, and a day of time to get started. You can use your own reporter or reporters provided in the Nucleus DNA collection.

• Materials
• Boot up PURE
• Boot up liposome encapsulation
• Encapsulate PURE
• References

Materials

Boot up PURE

PURExpress is a commercially available implementation of the PURE synthetic cytosol, provided by NEB. While the composition of PURExpress is proprietary, it’s sufficient to get stared with prototyping.

1. Use PURExpress to express the included DHFR control template (if you can measure absorbance 340 nm). (Optionally): you can express fluorescent (e.g., GFP) or colormetric (e.g., cjBlue) reporters. See Assemble PURE Reactions for more details.
2. Following Measuring Fluorescence with Plate Reader, measure absorbance (340 nm for DHFR; adjust if using your own colormetric reporter) or fluorescence (i.e., if expressing a fluorescent reporter; look up your reporter’s emission and excitation peaks and adjust your plate reader accordingly).

And if you’ve gotten to this step, then congratulations! PURE is the most commonly used implementation for synthetic cytosols, and you now know how to assemble and execute a PURE reaction. Well done!

Boot up liposome encapsulation

Let’s make a simple synthetic membrane. Here we will encapsulate a very simple cytosol (i.e., buffer plus a fluorescent dye). See Buffer-in-Buffer Liposomes for more details.

1. Make inner buffer (sucrose plus HPTS) and outer buffer (glucose): Inner and Outer Solutions.
2. Follow Buffer-in-Buffer Liposomes protocol.
3. Measure liposomes with Light Microscopy of Liposomes
4. Analyze results with Light Microscopy Analysis

Encapsulate PURE

You’ve now made cytosols and membranes separately. The next step is to combine the two:

1. Assemble PURExpress reactions as you did above (see Assemble PURE Reactions for more details).
2. Encapsulate assembled PURE reactions in a liposome using
3. Measure liposomes with Light Microscopy of Liposomes
4. Analyze results with Light Microscopy Analysis

References

PURExpress - commercially available PURE (NEB)