Researchers open up a potential new route for designing synthetic cells

The Dutch and American teams have developed a cell-free expression system based on a minimal cell.

Essential for the construction of synthetic cells, cell-free expression (CFE) systems use the transcription and translation machinery of living cells to produce proteins in vitroWilhelm Huck’s research team, in collaboration with the Adamala Lab and the Craig Venter Institute, have developed a CFE system based on a minimal synthetic bacterium. Their work opens up a possible new path for the design of synthetic cells. The results were published in ACS Synthetic Biology in June 2023.

Wilhelm Huck

Building synthetic cells from the bottom up, i.e. from scratch, could both unlock the mystery of how living systems work and contribute to the development of new technologies with a positive impact on the environment and our health. The first step in this exciting challenge is to build a cell-free expression (CFE) system, a simple system that contains the ingredients needed to express genes and produce proteins, which perform almost all the tasks required for organisms to function properly.

A minimal synthetic bacterium to develop a new CFE platform

Researchers usually develop CFE systems by first isolating the lysate of living cells, a mixture of intracellular materials needed to produce proteins, and then reassembling them in vitro. Efficient and well-established CFE systems come from E. coli, a bacterium with 4401 genes. But building a synthetic cell able of producing all the E. coli proteins remains extremely difficult. The Huck group at Radboud University Nijmegen in the Netherlands, therefore set out to design a CFE system from a minimal synthetic bacterium.

“The J. Craig Venter Institute has minimised the genome of Mycoplasma mycoides into a near minimal bacterial strain called Syn3A. Syn3A has 493 genes, making it an interesting model for the bottom-up construction of a synthetic cell,” explained Wilhelm Huck.

The project lasted over four years and proved to be much more arduous than anticipated. The team published the results in ACS Synthetic Biology in June 2023.

Extracting the cell lysate appears almost impossible

“Syn3A is a parasite. Its genome is very small because it develops inside other cells and takes the building blocks of the existing cell by degrading RNA using nuclease and protease enzymes. This complicated our project because the nuclease and protease were so active that the ribosomes, RNA and DNA that we needed to extract from our lysate to design the CFE system, were degraded in a matter of minutes.”

For this project, the Huck group collaborated with the Adamala Lab at the University of Minnesota in the United States. “This collaboration was essential to maintain the momentum and motivation needed to take on this major challenge. We had many transatlantic conversations to share our knowledge of what worked and what didn’t, and to brainstorm new approaches to solving this problem. Finally, my PhD student Andrei Sakai made a breakthrough by trying the nitrogen decompression method,” said Wilhelm.

The nitrogen bomb, a method to stop the action of nuclease and protease enzymes

ACS Synth. Biol. 2023, 12, 6, 1616-1623

Andrei, the first author of the article, subjected the Syn3A cell to a very high nitrogen pressure, before suddenly releasing the pressure to make the cell explode. Neither the nuclease nor protease enzymes had time to degrade the other components and the lysate could be extracted to build the CFE system. The team then used active machine learning tools to optimise the composition of the Syn3A CFE system.

“Thanks to Andrei’s tenacity, we have managed to build a CFE platform based on a minimal cell, and this is a starting point for exploring more complex experiments.”

Next steps: improving the robustness of the system and expressing more Syn3A proteins

The Syn3A-based CFE system currently produces one protein from the Syn3A bacterium. The group would like to increase the robustness of the system and express all the proteins of Syn3A, the first step towards building a synthetic cell.

More information:

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium
Andrei Sakai, Aafke J. Jonker, Frank H. T. Nelissen, Evan M. Kalb, Bob van Sluijs, Hans A. Heus, Katarzyna P. Adamala, John I. Glass, and Wilhelm T. S. Huck
ACS Synthetic Biology 2023 12 (6), 1616-1623, DOI: 10.1021/acssynbio.3c00114


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