Synthetic biology: Bottom-up meets top-down

We used proton gradients from top-down engineered light-harvesting E. coli to actuate DNA origami nanostructures. The DNA origami, in turn, controls the deformation of bottom-up assembled synthetic cells.

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Live as we know it arises from the complex interplay of its functional parts, leading to emergent properties that distinguish living from non-living matter or biology from chemistry and physics. Attempts to engineer cells from scratch, as perused in the field of bottom-up synthetic biology, should therefore focus on system integration as much as on the development of new functional parts [1, 2]. 


It is possible that a strategic merger of top-down and bottom-up synthetic biology may be helpful to accelerate progress towards a synthetic cell. It may circumvent challenges related to protein purification and reconstitution while capitalizing on the control and tunability that characterize bottom-up assembled systems. So we set out to find a common language for top-down and bottom-up engineered synthetic cells.

Solving the puzzle of life: Synthetic biology has long focused on functional modules.
Now it is time to bring the pieces of the puzzle together. This may lead to a synthetic cell that is
more than the sum of its parts.

Proton gradients are the universal form of energy generation and storage in living systems. Maybe that is a good starting point! It is known that DNA nanostructures can be actuated with proton gradients by making use of the DNA triplex motif – a nanomechanical switch that responds to pH changes in the environment [3]. We thus equipped a DNA origami plate with the triplex motif. At acidic pH, the DNA origami is inactive. At basic pH, the triplex opens up such that the DNA origami can bind to DNA-functionalized giant unilamellar lipid vesicles. Polymerization of the DNA origami leads to vesicle deformation [4]. 


But where do the proton gradients come from? We engineered E. coli bacteria to overexpress the inward-proton-pump xenorhodopsin [5]. These light-harvesting E. coli can now reversibly switch the pH of their environment: Illumination increases the pH, while protons flow back along the concentration gradient in periods of darkness. 


The integration of the DNA origami with the triplex motif, the lipid vesicles and the E. coli took us on a scientific detour. We realized that the triplex motif was no longer operating as designed. We soon realized that this was because we had changed one of the fluorophores on the DNA-functionalized vesicles, which completely altered the kinetics as well as the steady state of the triplex attachment. In a sperate work, we show with experimenents and MD siumulation how knowledge about the effect of DNA modifications on dynamic DNA nanostructures can be harnessed to tune their energy landscapes [6]. 


Only with this knowledge, the system integration was finally successful: We have finally realized a quite complex reaction pathway: When we turn on the light, the proton pumps in the E. coli become active, the pH changes, which, in turn, is activating the DNA origami. The DNA origami attaches to the membrane of a lipid vesicle, where it polymerizes to deform the synthetic cell. The bacteria thus communicate with the synthetic cells to adapt their shape. 


Ultimately, we hope that the realization of complex reaction pathways and the integration of functional modules may lead to synthetic cells with emergent properties. One day, a synthetic cell may be more than the sum of its parts.

References:

[1] Göpfrich, K., Platzman, I., & Spatz, J. P. Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells. Trends in Biotechnology 36, 938–951 (2018).

[2] Schwille, P. et al. MaxSynBio: Avenues Towards Creating Cells from the Bottom Up. Angew. Chemie Int. Ed. 57, 13382–13392 (2018). 

[3] Green, L., Amodio, A., Subramanian, H. K. K., Ricci, F., & Franco, E. pH-driven reversible self-assembly of micron-scale DNA scaffolds. Nano Letters 17, 7283–7288 (2017).

[4] Franquelim, H. G., Dietz, H., & Schwille, P. Reversible membrane deformations by straight DNA origami filaments. Soft Matter 17, 276–287 (2021).

[5] Ritzmann, N., Thoma, J., Hirschi, S., Kalbermatter, D., Fotiadis, D., & Müller, D. J. Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes. Biophysical Journal 113, 1–6 (2017).

[6] Jahnke, K., Grubmüller, H., Igaev, M., &  Göpfrich, K. (2021). Choice of fluorophore affects dynamic DNA nanostructures. Nucleic Acids Research 49, 4186–4195.

Kerstin Göpfrich

Dr., Max Planck Institute for Medical Research