A useful synthetic minimal cell.

1 Function

1. What would your synthetic cell do? What is the input and what is the output.
   

   Release compounds on a schedule within a biological system via tunable oscillator, by synthesizing compounds in a sub-compartment and releasing synthetic Outer Membrane Vesicles containing the delivery compound.

   Input: light-tuneable oscillator, glucose Output of the SMC: selective liposome destroying enzymes Output of the whole system: medications, on schedule tunable oscillator: <reference> selective liposome destruction: <reference>

2. Could this function be realized by cell free Tx/Tl alone, without encapsulation?
   

   Yes, very inefficiently. This is how drug delivery currently works.

3. Could this function be realized by genetically modified natural cell?
   

   Yes, but more considerations for interactions between the medication to be delivered and the cell would need to be taken into account

4. Describe the desired outcome of your synthetic cell
   

   A liposome containing our drug and an enzyme which breaks down its wall

2 Components

1. Design all components that would need to be part of your synthetic cell.
   

   A repressilator A liposome containing the main cell An enzyme which builds sub-liposomes with stronger walls An enzyme which synthesises the drug to be delivered An enzyme which builds a protein which breaks down the cell wall of the subliposome

2. What would be the membrane made of?
   

   Phospholipids + cholesterol?

3. What would you encapsulate inside? Enzymes, small molecules.
   

   Cell free Tx/Tl system, genes for repressilator, genes which express proteins which become enzymes for sub-liposome synthesis, drug synthesis, and genes which express proteins for enzymes that selectively break cell walls

4. Which organism your tx/tl system will come from? is bacterial OK, or do you need mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian).
   

   Bacterial, as far as I know.

5. How will your synthetic cell communicate with the environment? (hints: are substrates permeable? or do you need to express membrane channel?)
   

   The outer membrane should be permeable to glucose, application should involve a large cache of feed to synthesize the appropriate drugs from. It should be possible to suppress the oscillator externally via light, as a debugging technique.

3 Experimental details

1. List all lipids and genes (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick actual gene)
   

   Lipids: POPC, cholesterol Enzymes: bacterial cell free tx/tl, selective-cell-wall-breaking-enzyme, drug synthesis (let's pick aspirin, maybe?) Genes: <no idea> Biological cells: <no idea>

2. How will you measure the function of your system?
   

   Check drug concentrations in solution after tuning oscillators to different frequencies.

4 Scheme