By: TUDelft/TNW/Communication
Andrew Scott, working at the Christophe Danelon Lab of TU Delft, has taken an important step in the effort to create a synthetic cell. Scott has reconstituted the protein machinery to synthesize phospholipids, the molecules that make up cell membranes.

Creating life by reverse engineering the biological cell. That, in a nutshell, is the goal of the Christophe Danelon Lab. The research group is taking a ‘global’ approach to this grand challenge, trying to reconstruct every essential cellular function. Ambitious? It certainly is. But the researchers are proving that their approach may very well be the way to go. The latest breakthrough coming out of the Danelon Lab is the result of a study carried out by Andrew Scott. After 4.5 years of PhD research, Scott has managed to synthesize phospholipids, the molecules that make up the basic structure of membranes.

Layer of lipids
Membranes are important elements of cells. They function as barriers, shielding the interior of a cell from the outside environment. They can also be found within the cell, surrounding the various organelles, the ‘little organs’ that execute the basic cellular functions. The fundamental structure making up membranes is called the ‘phospholipid bilayer’, which is only a few nanometers thick and consists of two layers of lipid molecules. These molecules, in turn, are composed of a water-loving (or ‘hydrophilic’) head and a water-fearing (or ‘hydrophobic’) tail. When brought into contact with water, the lipids spontaneously assemble to form a sealed compartment, much like a Macedonian phalanx.



It starts with genes
Phospholipid molecules are created through a series of complex chemical reactions that are set in motion by specialized proteins (enzymes). Starting with DNA containing the genes encoding for these enzymes, a mixture of compounds that convert the DNA program into protein and simple ‘precursor’ molecules of phospholipid, Scott managed to copy these reactions in a test tube. The DNA he used was originally extracted from E. coli, a relatively simple and well-understood class of bacteria that can be found in the human gut. ‘The DNA was then purified, undergoing multiple molecular biological steps in order to end up as the final DNA template that we could use’, says Danelon.

In the same way that a series of commands can boot up a computer program, the DNA reading started the cascade of biochemical reactions that ended with the production of phospholipid molecules. ‘So we synthesized not only the lipids, but also the chain of proteins involved in this pathway’ explains Danelon.

Interestingly, producing the lipids was not the most difficult part of this research project. ‘The biggest challenge was developing a significantly sensitive method to detect the phospholipids’, says Scott. ‘I struggled for a year and half with various techniques that did not have the necessary resolution.‘

In the end, the arrival of an mass spectrometer made it possible to detect tiny amounts of lipids. ‘Once we were able to detect these lipids, exploring the functionalities of the various enzymes was fairly simple and fruitful.‘

Combining the parts
So far, the Danelon Lab has reverse engineered several key cellular mechanisms. An important next step will be the optimization of all of these different modules. The amount of lipids that was produced in this study, for instance, was quite modest. This means that the compartments they form will not be able to grow and divide, as normal cells are wont to do.

Looking even further ahead, the challenge the Danelon group faces is combining the various biological modules into one living, self-replicating cell. Most likely, merely putting the parts together will not be enough, because the processes that are to make up the minimal cell as envisaged by the Danelon Lab require a mere 150 genes. However, the simplest life form known so far is an artificial bacterial cell created in the Craig Venter Institute, which consists of 473 genes, quite a few more than 150. Moreover, it is not at all clear what the exact function of about twenty percent of these 473 genes is. This is something that also needs to be addressed in the future.     

As complex as a city
This much is obvious: there is still a lot of work to do. When asked if he believes creating a synthetic cell is even possible, Scott answers that he does. ‘But I think we should expect such a cell to be at least as complex as a modern city, meaning that no individual will understand all the parts’, he says. So how much longer do we have to wait? ‘I’m afraid that’s impossible to say given our current knowledge of biology, says Danelon. ‘But one thing is for sure: this project will keep us busy for quite some time to come.’

Article
Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes by Andrew Scott, Marek J. Noga, Paul de Graaf, Ilja Westerlaken, Esengul Yildirim and Christophe Danelon was published in Plos One on 6 October 2016.