NANOCELL is part of the Eurocores program Synthetic Biology: Engineering Complex Biological Systems (EuroSYNBIO)
The mission of NANOCELL is to engineer biomimetic molecular machineries of the cell as building blocks that can be robustly and flexibly assembled to nanocells with controllable functionality not found in nature. To approach this goal, we will take nature’s cellular machineries apart and explore their potential to reconstitute them in new ways. The synthetic ‘NANOCELL’ resembling a molecular factory is one strong vision that drives the project. In its first step NANOCELL will master the control of the following biomolecular machines developed by nature, (i) F1Fo-ATP synthases, (ii) ATP-driven nucleic acid translocating machines, (iii) ATP synthase based propellers, (iv) proton-driven drug, solute and peptide transporters, and (v) spectrally tuned light-driven proton pumps (Figure 1). Most of these machines have in common that either their structure and mechanism and/or their function have been characterized to unprecedented accuracy very recently, which bears the chance to move on now to this engineering approach. From a synthetic biology approach we will reconstitute these machines into stable synthetic vesicles. Proton gradients that either power biomolecular machines will be generated by spectrally tunable light-driven proton pumps bacterio- and proteorhodopsins. Short-term goals are to develop strategies to manipulate and engineer the individual biomolecular machines to be used as building blocks to establish a NANOCELL. Procedures for their reconstitution into synthetic vesicles building the frame of the future NANOCELL will be established. In the long-term, we intend to use several of these engineered building blocks to create complex NANOCELLS. With this approach of establishing engineered building blocks we can functionalize NANOCELLS to generate, for example, a proton-gradient that guides the uptake or release of drugs, peptides, DNA, or solutes or to physically move the NANOCELL. It is also thought to spectrally tune proton-gradients to synthesize ATP used for minimal metabolic processes within the NANOCELL.
Using bacterio- and proteorhodopsins, light of different wavelengths can be used to create and adjust an H+-gradient across the synthetic NANOCELL membrane. Depending on the direction of X-rhodopsin insertion, the H+-gradient can be increased or reversed. The H+-gradient is used to release or uptake molecular compounds by H+-driven transporters (co-transporter or antiporter). An H+-driven DNA translocating motor will allow uptake and release of DNA. We use H+- and Na+-driven ATP synthases for ATP synthesis and NANOCELL motility. F-ATP synthases use the H+-gradient to synthesize ATP that can be used for minimal metabolic systems hosted in the interior of the NANOCELL. Manipulated F-ATP synthases areused as Na+-driven (alternatively H+-driven) engine that rotates a propeller toactively move the NANOCELL.
Daniel Müller, Department for Biosystems Science and Engineering, ETH Zürich, Switzerland
Helmut Grubmüller, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany
Thomas Meier, Max-Planck-Institute of Biophysics, Frankfurt, Germany
Richard Berry, Physics, Oxford University, UK
Wolfgang Meier, Chemistry, University Basel, Switzerland
Dimitrios Fotiadis, Institute of Biochemistry and Molecular Medicine, University Bern, Switzerland
Sven Panke, Department for Biosystems Science and Engineering, ETH Zürich, Switzerland
Associate partner: Jose Carrascosa, Centro Nacional de Biotecnologia, Madrid, Spain
The set of biomolecular machines aimed in exploiting within NANOCELL set a unique stage to implement rudimentary aspects of cellular metabolism. One central element of cellular metabolism is protein production. Protein production is one of the most energy intensive cellular processes. This has frustrated various attempts at implementing highly efficient in vitro protein production systems, as regeneration of the energy carrier, ATP, required the addition of chemical compounds to re-synthesize ATP from ADP. As a consequence, the protein production process stopped either when degradation products accumulated to a sufficient extent or when the enzymatic machinery required for ATP regeneration was no longer intact due to protein degradation or aging. In contrast, the combination of membrane-bound light-driven proton pumps and ATP synthases suggests an opportunity to regenerate ATP in an orthogonal fashion which does not contribute to the degeneration of protein production. Furthermore, the possibility to compartmentalize the protein production in stable synthetic (i.e. coblock polymer) vesicles presents unique opportunities to exploit biological evolution processes for enzyme development (Walser 2008 & 2009). Currently, the leading protein evolution systems use emulsion technologies. The requirements of preparing “monoclonal” droplets by mixing two bulk phases (one aqueous and one oil phase) lead inevitably to a major proportion of droplets without DNA. The integration of a viral DNA motor into the vesicle-system might open a road to an elegant solution to the problem of recruiting one DNA molecule into one vesicle. Therefore, we want to reconstitute a rudimentary and ultimately light-driven protein production metabolism in stable vesicles and to combine this with the viral-DNA motor as a tool to import single DNA molecules from a population of DNA variants.
Walser, M, Leibundgut, Panke, S, Held, M (2008). Isolation of monoclonal microcarriers colonized by fluorescent E. coli. Cytometry A: 73A: 788-798.
Walser, M, Pellaux, R, Meyer, AJ, Bechtold, M, Vanderschuren, H, Reinhardt, R, Panke, S, Held, M (2009). Looking for a needle in a haystack: high throughput colony PCR screening in nanoliter-reactors. Nucleic Acids Res 37: e57.
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