Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell

Artificial systems capable of self-sustained movement with self-sufficient energy are of high interest with respect to the development of many challenging applications, including medical treatments, but also technical applications. The bottom-up assembly of such systems in the context of synthetic biology is still a challenging task. In this work, we demonstrate the biocompatibility and efficiency of an artificial light-driven energy module and a motility functional unit by integrating light-switchable photosynthetic vesicles with demembranated flagella. The flagellar propulsion is coupled to the beating frequency, and dynamic ATP synthesis in response to illumination allows us to control beating frequency of flagella in a light-dependent manner. In addition, we verified the functionality of light-powered synthetic vesicles in in vitro motility assays by encapsulating microtubules assembled with force-generating kinesin-1 motors and the energy module to investigate the dynamics of a contractile filamentous network in cell-like compartments by optical stimulation. Integration of this photosynthetic system with various biological building blocks such as cytoskeletal filaments and molecular motors may contribute to the bottom-up synthesis of artificial cells that are able to undergo motor-driven morphological deformations and exhibit directional motion in a light-controllable fashion.R.A., V.N., E.B., I.G., and A.G. acknowledge support from the European Union’s Horizon 2020 research and innovation programme under grant agreement MAMI No. 766007. C.K., A.B., E.B., K.S., I.G., T.V.K., and A.G. thank MaxSynBio Consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Societ

Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions

International audienceSelf-sustained metabolic pathways in microcompartments are the cornerstone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made micro-compartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells

Cooperative Binding of PhoB^DBD to Its Cognate DNA SequenceA Combined Application of Single-Molecule and Ensemble Methods

A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein–DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB^DBD). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB^DBD and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB^DBD, semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein–DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the <i>pho</i> box by the protein PhoB^DBD could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process

Cooperative Binding of PhoB(DBD) to Its Cognate DNA Sequence-A Combined Application of Single-Molecule and Ensemble Methods

Ritzefeld M, Walhorn V, Kleineberg C, et al. Cooperative Binding of PhoB(DBD) to Its Cognate DNA Sequence-A Combined Application of Single-Molecule and Ensemble Methods. Biochemistry. 2013;52(46):8177-8186.A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process