In the area of synthetic biology, a "living" artificial cell has been defined as a completely synthetically made cell that can capture the energy, maintain ion gradients, contain macromolecules as well as store information and have the ability to mutate. Although not completely artificial because the cytoplasmic components, as well as the membrane from the host cell are kept, the engineered cell is under control of a synthetic genome and is able to replicate.

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1) Pick a function for your synthetic minimal cell.​

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

The synthetic cell is used as an RNA thermometer to produce GFP (Green Fluorescent Protein) when the temperature rises.

 

“All constructs were transformed into E. coli and tested for temperature responsiveness of GFP expression to assess their possible functionality as RNA thermometer. In the paper, the bacterial strains were grown at four different physiological temperatures: 17°C, 22°C, 30°C and 37°C followed by protein extraction and determination of GFP protein amounts by western blotting.” (source 3)

 

Input: RNA thermometers (RNATs)

Output: GFP produced in bacteria

 

1B) Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

Yes, the goal is to create a cell which changes colour based on temperature change. Since the product does not directly need to interact with other/natural cells to do this, encapsulation is not needed. The other main goal of encapsulation is to protect the integrity of the interior of the cell by allowing certain substances into the cell while keeping other substances out. But since we control what is in the cell, and what its environment looks like this will not be needed.

 

1C) Could this function be realized by genetically modified natural cell?

Yes, the RNA thermometer could be incorporated into a transformed gene.

 

1D) Describe the desired outcome of your synthetic cell operation.

If the temperature (T) rises, the synthetic cell expresses Green Fluorescent Protein (GFP).

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“RNA thermometers are RNA-based genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a conformation that masks the ribosome binding site [Shine–Dalgarno (SD) sequence] within the 5′-untranslated region (5′-UTR) and, in this way, prevents ribosome binding and translation. At elevated temperatures, the RNA secondary structure melts locally, thereby giving the ribosomes access to the ribosome binding site to initiate translation. RNA thermometers differ from riboswitches (1) in that they do not require binding of a ligand (metabolite) to induce the conformational change, but instead, directly respond to temperature.” (Neupert, Karcher & Bock, 2008). 

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