SYNTHETIC MINIMAL CELL

A minimal cell is an engineered particle that mimics one or many functions of a biological cell. The term refers rather to the idea that certain functions or structures of biological cells can be replaced or supplemented with a synthetic entity.

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.

1) Pick a function.

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

Sources:

Juliane Neupert & Ralph Bock (2009). Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria. Nature. doi:10.1038/nprot.2009.112 http://www.nature.com.ezp-prod1.hul.harvard.edu/nprot/journal/v4/n9/pdf/nprot.2009.112.pdf
Juliane Neupert, Daniel Karcher, and Ralph Bock (2008). Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic Acid Research. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2577334/
Zachary Z. Sun, Clarmyra A. Hayes, Jonghyeon Shin, Filippo Caschera, Richard M. Murray, and Vincent Noireaux (2013). Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Journal of Visualized Experiments. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960857/
C. Eric Hodgman and Michael C. Jewett (2011). Cell-Free Synthetic Biology: Thinking Outside the Cell. Metabolic Engineering. doi: 10.1016/j.ymben.2011.09.002 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322310/

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).

“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.” (source 2)

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

2A) What would be the membrane made of?

As told in question 1B, the synthetic cell will not be interacting with other cells. It also will have no harm to the environment around it, since that is controllable. The only reason a membrane might be needed is when particular research on the cooperation between organelles in a cell is needed, in this case, a droplet will be sufficient as cell boundary.

2B) What would you encapsulate inside?

Cell-free Tx/Tl System, SD sequences, ASD sequences, restriction enzymes, reporter gen

Most of the other small molecules like ribosomes and mRNA are already in the ECE.

source: an online course, writer unknown http://nptel.ac.in/courses/102101040/modules/module4/lec24/1.5.html

2C) Which organism your TX/TL system will come from?

Bacterial, the Escherichia coli extract (ECE).

2D) How will your synthetic cell communicate with the environment?

This synthetic cell is capable of detecting and responding to a change of temperature in its environment which enables a dynamic modulation of gene expression through diverse mechanisms in order to express GFP in presence of high temperatures.

3) Experimental details

3A) List all lipids and genes

-RNA thermometers (RNATs)

-Bacterial strain and vectors:

E.coli
pBluescriptII SK(+) cloning plasmid vector

-Enzymes and antibodies:

Anti-mouse IgG peroxidase conjugate
Monoclonal anti-GFP antibody

-Restriction endonucleases:

BamHI

NcoI

RNase A

T4 DNA ligase

Full list of reagents can be found in the protocol (http://www.nature.com.ezp-prod1.hul.harvard.edu/nprot/journal/v4/n9/pdf/nprot.2009.112.pdf)

3B) How will you measure the function of your system?

The GFP output of the cells can be measured by comparing the colours.

Scheme (bonus)

This picture represents the workflow design, construction and experimental testing of synthetic RNA thermometer. RE represents unique restriction sites for cloning.