What aspect does a system require to be called alive? Discussed by Schrödinger’s book "What is Life?" and further cultivated perhaps most prominently by the synthetic cell community, a combination of DNA for replication, metabolism, and information processing together with a membrane to hold everything together appear as crucial parts. However, the necessity for some of those properties is questionable when it comes to building synthetic systems. Thermal gradients, for instance, accumulate molecules independent of cellular compartments (i.e., membranes) but driven through constant energy dissipation⁽¹⁾. Because of these fuzzy boundaries, most scientists do not specify how synthetic living systems would look like in the future: it depends on context.

Our lab (Bar-Ziv) pioneered on-chip compartments programmed with DNA molecules as solid-state artificial cells^(2–5). Simple geometries define how long proteins stay inside the compartment before being diluted out through a thin capillary. The genetic programs rely on RNA polymerases, ribosomes, and other ingredients for cell-free gene expression, which are extracted from E. coli cells^(6,7). Throughout the years, we worked on a range of systems, including genetic circuits with transcription factors, DNA modifications with recombination proteins⁽⁸⁾, and machine assemblies with massive parts of the T4 virus⁽⁹⁾ and ribosome^(10,11). Inside our cell-sized compartments, large numbers of DNA molecules immobilized in dense brushes promote high localized concentrations of genetic products. This increases the chances that molecules bump into each other and form desired higher-order complexes.

We set out to test the effects of local concentrations by studying the stability of genetic decision-making in on-chip artificial cells⁽⁵⁾. We cloned part of the lambda phage genetic toggle switch into DNA molecules and loaded the compartment with DNA at very high concentrations. We established its expected switching response and went down by six orders of magnitude, drastically reducing the concentration of DNA and proteins. Around 20 DNA molecules, each producing tens of proteins per hour in a compartment that is ~1000-fold larger than a bacterium. Against our expectations, we found genetic regulation to occur even in these dilute conditions. It was fuzzy compared to the switching at high concentrations. However, there still, we found strong indications that transcription factors bind to the DNA to regulate gene expression. How could that work at such minute concentrations?

Two decades ago, the Mirny lab put forward a possible explanation⁽¹²⁾. They proposed that coupled transcription and translation could transiently tether the transcription factors to the DNA and help find their target sites nearby on the chromosome. In living cells like E. coli, most transcription factors’ genes and target sites are positioned very close along the genome. Likewise, Nirenberg visualized the coupling of transcription and translation in a frozen state during the 60s⁽¹³⁾. But visualization of the dynamic and short-lived synthesis of genetically encoded proteins on the DNA remained a challenge. Tools to study either transcription or translation separately exist, but the entire process, from the RNA polymerase making mRNA from DNA to the ribosome finding the mRNA, and producing the protein, which can happen within minutes, required a new approach.

We asked Kai Johnsson and Nicolas Lardon at the MPI for Medical Research in Germany to provide us with some of their newly developed fluorogenic dyes, reacting with HaloTag proteins. This fluorescent reporting system enables rapid imaging of nascent HaloTag proteins directly on the DNA, much faster than the slow-maturating fluorescent proteins. We performed many control experiments to exclude the notorious issues with single-molecule imaging, e.g., unspecific adsorption and fluorescent background signals. This time, inside a simple microfluidic flow channel without confinement by on-chip artificial cells, we watched our single-molecule microscope and found blips of protein signals appearing on dilute spots of DNA molecules. We observed the emergence of individual proteins from their genes tethered through transient complexes of RNA polymerases, messenger RNAs, and ribosomes. Heureka!

With our new reporter system, we could finally verify our hypothesis: nascent transcription factors can regulate downstream genes on the same DNA molecule. We sought to test the prospects by encoding a full genetic circuit with an activator and repressor. The activator increased its local concentration to initiate the repressor synthesis that would shut down the activator production. The circuit started pulsating in dilute cell-free conditions, driven by only a handful of proteins transiently tethered to DNA by coupled transcription and translation complexes. The efficiency and accuracy can certainly be improved. Still, our findings present the smallest autonomous genetic circuit to date⁽¹⁴⁾. In the context of synthetic cell systems, our results seem to soften the requirements for cellular compartments, at least in the first stages of their development.

Calling a genetic circuit alive would be too much, but perhaps the reconstitution with single molecules pursues the most basic, unmasked, and simple representation of biological processes. At least, we hope that it provides an exciting journey toward constructing fully autonomous synthetic life, whatever shapes and looks it will have.

The authors of this blog post are F. Greiss, S.S. Daube, V. Noireaux, and R. Bar-Ziv.

 References

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