Nature Chemistry 11, 32–39 (2019)
Signalling and differentiation in emulsion-based multi-compartmentalized in vitro gene circuits

Aurore Dupin and Friedrich C. Simmel

Abstract
Multicellularity enables the growth of complex life forms as it allows for the specialization of cell types, differentiation and large-scale spatial organization. In a similar way, modular construction of synthetic multicellular systems will lead to dynamic biomimetic materials that can respond to their environment in complex ways. To achieve this goal, artificial cellular communication and developmental programs still have to be established. Here, we create geometrically controlled spatial arrangements of emulsion-based artificial cellular compartments containing synthetic in vitro gene circuitry, separated by lipid bilayer membranes. We quantitatively determine the membrane pore-dependent response of the circuits to artificial morphogen gradients, which are established via diffusion from dedicated organizer cells. Utilizing different types of feedforward and feedback in vitro gene circuits, we then implement artificial signalling and differentiation processes, demonstrating the potential for the realization of complex spatiotemporal dynamics in artificial multicellular systems.
Full text  https://doi.org/10.1038/s41557-018-0174-9

ACS Synthetic Biology 8, 148–157 (2019)
Stationary patterns in a two-protein reaction-diffusion system

Philipp Glock, Beatrice Ramm, Tamara Heermann, Simon Kretschmer, Jakob Schweizer, Jonas Mücksch, Gökberk Alagöz and Petra Schwille

Abstract
Patterns formed by reaction-diffusion mechanisms are crucial for the development or sustenance of most organisms in nature. Patterns include dynamic waves, but are more often found as static distributions, such as animal skin patterns. Yet, a simplistic biological model system to reproduce and quantitatively investigate static reaction-diffusion patterns has been missing so far. Here, we demonstrate that the Escherichia coli Min system, known for its oscillatory behavior between the cell poles, is under certain conditions capable of transitioning to quasi-stationary protein distributions on membranes closely resembling Turing patterns. We systematically titrated both proteins, MinD and MinE, and found that removing all purification tags and linkers from the N-terminus of MinE was critical for static patterns to occur. At small bulk heights, dynamic patterns dominate, such as in rod-shaped microcompartments. We see implications of this work for studying pattern formation in general, but also for creating artificial gradients as downstream cues in synthetic biology applications.
Full text  https://doi.org/10.1021/acssynbio.8b00415

Nano Letters 18, 7133–7140 (2018)
Light-induced printing of protein structures on membranes in vitro

Haiyang Jia, Lei Kai, Michael Heymann, Daniela A. García-Soriano, Tobias Härtel and Petra Schwille

Abstract
Reconstituting functional modules of biological systems in vitro is an important yet challenging goal of bottom-up synthetic biology, in particular with respect to their precise spatiotemporal regulation. One of the most desirable external control parameters for the engineering of biological systems is visible light, owing to its specificity and ease of defined application in space and time. Here we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on time scales of seconds, and the pattern density can be precisely controlled by protein concentration, laser power, and activation time. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. Thus, light mediated sequential protein assembly in cell-free systems represents a promising approach to hierarchically building up the next level of complexity toward a minimal cell.
Full text  https://doi.org/10.1021/acs.nanolett.8b03187