Cell-Sized Mechanosensitive and Biosensing Compartment Programmed with DNA

Cell-free expression (CFE) has been recently reshaped to become a highly versatile technology applicable to an increasing number of research areas. The new generation of DNA-dependent cell-free transcription-translation systems (TXTL) has been engineered to address applications over a broad spectrum of engineering and fundamental disciplines, from synthetic biology to biophysics and chemistry. In particular, engineering artificial cells integrating active membrane functions is critical to develop mechanically robust compartments capable of sensing the physical and chemical environment. Phospholipid vesicles are particularly useful for encapsulating TXTL reaction in cell-sized compartments because lipid bilayers are the natural substrates for membrane proteins such as channels and receptors. Expressing membrane proteins is a critical step towards constructing synthetic cells with functional interfaces to provide, for instance, transport or catalytic capabilities to a vesicle. Mechanosensitive channels of large conductance (MscL) are membrane channels that are integral to bacterial survival. They respond to increased membrane tension and serve as an emergency release valve to equilibrate osmotic pressure during osmotic downshock. MscL from E. coli K12 strain was cloned into a vector capable of producing the protein in an E. coli cell-free TXTL system. MscL association with the lipid bilayer and its subsequent activation under hypo-osmotic shock were shown within cell-free encapsulated vesicles by monitoring the leakage of different-sized fluorescent dextran. In order to impart biosensing capability to the lipid vesicles, a genetically encoded calcium indicator (G-GECO) containing a GFP core fused with calmodulin and M13 (myosin light chain kinase) at its C and N terminals respectively, was also expressed in the cell-free system, and its calcium sensing capacity was demonstrated in bulk reactions. Further, the impermeable nature of the lipid bilayer to ions, was verified by the introduction of vesicles, encapsulated with cell-free expressed G-GECO, into a calcium rich solution. No fluorescence was observed for an extended period of time. However, the addition of an ionophore (A23187) resulted in a rapid jump in G-GECO intensity similar to that reported from bulk measurements. Simultaneous expression of MscL and G-GECO was then achieved inside phospholipid vesicles. These vesicles, when introduced into a calcium-containing iso-osmotic solution, did not show any increase in their luminal fluorescence. However, under hypo-osmotic conditions induced by an osmolarity mismatch of ~ 100mOsm/Kg, a significant increase in the fluorescence from G-GECO was observed indicating the influx of calcium ions from the outside solution into the encapsulated medium via the activated MscL channels. Thus, we have established the sensing capabilities of a genetically programmed artificial cell, subjected to simultaneous mechanical and chemical stimuli, which could be considered as the biological equivalent of an AND gate.