Abstract
In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.
Highlights
Functional encapsulation of biochemical reactions inside artificial biomimetic compartments is one of the most fundamental challenges in bottom-up synthetic biology
In the search for new materials for the fabrication of biomimetic and biocompatible compartments to encapsulate complex protein systems, we turned our attention to CYTOP, an amorphous fluorinated polymer that has good chemical resistance and ideal optical properties for imaging with standard fluorescence microscopy methods
We demonstrated that CYTOP chambers can be fabricated by two methods: one, by photolithography using reactive ion etcher (RIE), and second, by replica molding
Summary
Functional encapsulation of biochemical reactions inside artificial biomimetic compartments is one of the most fundamental challenges in bottom-up synthetic biology. Examples of preferred compartments include water-in-oil droplets and liposomes, which can be produced in large numbers and feature cell-sized volumes with phospholipid mono- or bilayer interfaces Through their mimicry of the cellular phospholipid interface, these compartments can support cytosolic reactions, and those that involve membranes.[1,2] On the other hand, deforming them to any other shape than their equilibrium spherical geometry remains a challenge,[3,4] and there is an ongoing quest to develop new platforms that support the custom design of reaction spaces other than spheres.[5,6] In this regard, the most prominent developments have been through microfabrication and photolithography techniques to pattern materials into any custom shape, including cell mimicries.[6−8] their effectiveness in biological studies depends greatly on the biocompatibility of the materials used to fabricate such structures.
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