NANOCELL

The overall goal of the NANOCELL consortium is to engineer biological components that can be assembled into artificial membrane-bound vesicles that have some of the functions of living cells. These NANOCELLS will be prototype biochemical factories for future applications in bio-nanotechnology. The membrane itself is the most important component. Another important component is F1FO (ATP-synthase). F1FO can be thought of as two rotary motors connected in opposition. Normally in nature the ion-driven FO generates more torque, and forces the ATP-driven F1 in reverse, thereby synthesizing ATP, the 'energy currency' of the cell. One goal of this part of the consortium (IP4) is to test and characterize the function of membranes containing F1FO in NANOCELLS, using fluorescence methods including single-NANOCELL fluorescence microscopy. F1FO and other membrane protein components will also be tested and characterized at the single-molecule level in droplet-on-hydrogel bilayers, which allow large planar membranes and membrane proteins that they contain to be observed with high-resolution optical microscopy. The final goal of IP4 is to use one or both of the F1FO rotary motors to drive a propeller that will allow NANOCELLS to move under their own power. The lab of IP4 is one of the world leaders in the study of the bacterial flagellar motor, which performs the same role in flagellated bacteria, and will use this expertise to guide the development of a swimming module for NANOCELLS.

The goal is to design and produce synthetic vesicles containing engineered variants of F1FO, in collaboration with other partners in the consortium. The two main contributions of IP 4 will be single molecule characterization of membranes and membrane proteins engineered for use as modules in NANOCELLS and a propulsion module to allow self-mobile NANOCELLS. 1) Testing the formation of ion gradients and membrane voltages by F1FO and light-driven ion pumps in vesicles. F1FO and light-driven ion pumps will be incorporated into vesicles by detergent purification and the formation of pH gradients due to H+ pumping will be quantified using fluorescent dyes. Bulk methods will test suspensions of vesicles, and microscopic methods will test single vesicles, allowing a characterization of heterogeneity and reliability. 2) Single-molecule diagnostics of membrane proteins. We will use the novel Droplet-on-Hydrogel Bilayer (DHB) technique to observe and characterize the function of individual membrane proteins. This will allow diagnosis of the effects of engineered modifications of the membrane proteins. 3) Self-mobile swimming NANOCELLS. We propose to use one or both of the F1FO rotary motors to drive a propeller that will allow NANOCELLS to move under their own power. The work will proceed in stages... 3a) Propellers based on bacterial flagellar filaments. We propose to borrow from nature the highly optimized solution evolved by flagellated bacteria, which swim driven by rotation of a rigid helical filament. Bacterial flagellar filaments well understood, cheap and easy to produce, and a range of genetic modifications with different helical forms can be obtained. 3b) Rotary motor based on F1FO. Efficient motors would be based on uncoupled motors, so that all of the free-energy of ion transport in FO or ATP hydrolysis in F1 would be available to drive the propeller, rather than driving the other, coupled molecular motor.

The long-range impact of NANOCELL is potentially vast. Synthetic biology promises to turn the extra-ordinary capabilities of the living world to technological ends, with applications in medicine, manufacturing and energy to name only those that spring first to mind. But we are still in the early days of synthetic biology. NANOCELL will test the feasibility of a components approach to systems biology (engineering biological molecules and their interactions, as opposed to the genetic approach of modifying the genomes of whole organisms). The project will demonstrate a prototype modular set of components and discover design principles and the strengths and weaknesses of different approaches. We expect NANOCELL to catalyze the emerging field of synthetic biology in two ways, first, by providing the technology to engineer biomolecular actuator systems, second, by providing and demonstrating a prototypic example for this synthetic biology approach. Accordingly, we expect the NANOCELL platform concept to catalyze the development of other synthetic biology platforms such as sensory, photosynthetic, biocomputing, molecular memory, biosynthetic, or bioenergetics platforms. The technical developments of IP4 will be widely applicable in the fields of membrane proteins and molecular motors. Use of the DHB method to study membrane proteins at the single molecule level is a new development with wide applicability, and we will help to advance the method. Characterization of F1FO at the single molecule level will inform the field of molecular motors, in particular casting light on how two molecular motors can be coupled together. A self-mobile artificial swimmer will be a dramatic example of the new capabilities of synthetic biology which will invigorate the field.