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August 27, 2004
Building Blocks for Biobots

"Biology today is at the same stage chemistry was a century ago — it's growing up quickly, making the transition from a largely descriptive discipline to one where we use what we know about biological systems to build new things," says Jan Liphardt, a Divisional Fellow in Berkeley Lab's Physical Biosciences Division (PBD) and a newly named assistant professor of physics at the University of California at Berkeley.

Someday biobots specialized for cleaning up toxic spills could analyze, seek out, and neutralize pollutants in a single application.

"In response to this development," Liphardt says, "PBD has established the nation's first Synthetic Biology Department," which is headed by PBD staff scientist Jay Keasling, a professor of chemical engineering at UC Berkeley. As founding members, Liphardt and his group are particularly interested in the design and construction of what Carlos Bustamante, head of PBD's Advanced Microscopies Department and a UCB professor of biochemistry, molecular biology, and physics, has dubbed "biobots" — autonomous, special-purpose robots, about the size of a virus or cell and composed of a small number of biological and artificial parts.

"One advantage of building biobots from the ground up" says Liphardt, "is that it's possible to use construction materials that are not normally found in biological systems."

Another advantage is that biobots — at least in their initial forms — will contain far too few components to replicate themselves, reducing any risk to the environment. "Since we still know only very little of how our biosphere works, it makes sense to proceed very cautiously," adds Derek Greenfield, a biophysics graduate student currently in the Keasling lab.

What to do with a biobot

Biobots have potential applications in medicine, national security, environmental protection, and many other fields. As one example, Liphardt envisions biobots designed to decontaminate toxic spills: "They could detect and identify specific hazardous chemicals, track down the extent of contamination, and internally manufacture whatever was needed to clean up the mess — all with one trip to the site."

That scenario may be some time off, but the knowledge gained in learning to build special-purpose synthetic devices will be immediately useful in analyzing and modeling the complicated dynamics of living cells. Much simpler and less versatile than highly evolved living systems, dedicated biobots will nevertheless mimic nature in important ways.

"Biobots will be able to assemble themselves from externally provided components; outwardly they'll consist of enclosures resembling cell walls or viral capsids," Liphardt says. "Molecular motors like flagella will give them motility. They'll have modules for using light or chemicals from their surroundings to make ATP" — small molecules cells use to store and transport energy — "and modules for sensing their environment and for performing specialized tasks."

Liphardt says, "Essentially, we wish to develop a collection of functional and structural LEGO blocks that we can mix and match. These building blocks will need to be robust, and they will have to be able to perform in concert with other building blocks, with no mutual interference. Achieving module separability is one of the biggest problems in this new field — ideally you would like to be able to combine, say, a motor with a sensor and a power source, without having to reengineer each of those modules every time you add another module, or subtly change one of them."

Biobots may be made from a set of standard elements chosen for the task at hand, including enclosures, motors, sensors, and energy producers.

Still, the task of building biobots is much simpler that trying to develop a self-replicating cell from scratch. Liphardt compares the first biobot to the Wright brother's first powered airplane: "The Wrights didn't have to reproduce the flight of birds in all its details," Liphardt says. "It was important for them to know that it could be done, and it was up to them to figure out what the essential physical principles were, and then build a machine out of the available materials."

Likewise, the scientists in Berkeley Lab's Synthetic Biology Department will mix and match various materials, some biological, like proteins, lipids, and DNA — and some, where needed, artificial, like silicon nitride or cadmium selenide.

Enclosures, for example, can be far less complex than real cell membranes. One kind of biobot enclosure might be self-assembled from proteins, while another biobot might get by with something as simple as a lipid bilayer. In the right environment, lipid molecules with hydrophilic (water loving) heads and hydrophobic (water fearing) tails readily self-assemble, heads out and tails in, in a tough, double-layered skin.

One of the early lessons of synthetic biology research is that living organisms can produce an amazing diversity of materials, ranging from nanoparticles of silver (made by certain bacteria) to single-mode optical fibers. Researchers at Bell Labs recently discovered that the deep-sea sponge Euplectella grows spiny spicula, which are optical fibers with excellent optical properties and better crack resistance than conventional, man-made fibers.

A biobotic tool kit

Constructing biobots will require special tools; Liphardt and his group are working to assemble the tool kit. One need is for a nanoscale analog of the industrial crane. "The established tools are optical tweezers, magnetic tweezers, and atomic force microscopes," he says, "but typically they are not used simultaneously." Imagine trying to build something and having to choose between being able to move girders around without knowing precisely where they are, or knowing exactly where they are but not being able to grab them. This is the current situation in the single-molecule field, and one of the most pressing tasks is to combine various forms of imaging and manipulations tools.

Combine the "crane" of optical tweezers with high-resolution imaging tools such as Fluorescence Resonance Energy Transfer (FRET), which can be used as a molecular ruler, and the construction of complex, nanometer-sized objects becomes more practical. In FRET, a molecule of fluorescent dye excited by incident light transfers its increased energy to an adjacent dye molecule; fluorescence decreases in the first molecule and increases in the second, a measure of the distance between them.

Jan Liphardt (left) and graduate student Hari Shroff with a recently built instrument combining optical tweezers with single-molecule fluorescence detection.

"We have recently built a combined optical tweezers/single molecule fluorescence instrument," Liphardt says, and his group has successfully used the new instrument to characterize nanoscale strain gauges based on conventional fluorescent dyes; it is designed to measure displacements and forces inside molecular machines. Fluorescent dyes can be specially designed or harvested from nature to bind at specific sites on target molecules. This makes it possible to optically measure — by changes in color or brightness — the forces involved in manipulating biological molecules, or to watch the mechanochemical processes inside living cells.

Thermodynamics on the nanoscale

Beyond the practical problems of assembling molecule-sized living machines lie daunting theoretical difficulties. Chemists and biologists are accustomed to describing material properties in terms of bulk averages. "Our intuition fails when applied to very small systems," Liphardt says. "Imagine a world where your car moves forward only on average, but every once in a while jumps backwards!"

Liphardt and his colleagues are using single-molecule techniques to refine and extend thermodynamic explanations, well suited to describing matter and energy in bulk, to the behavior of nanoscale devices. Much of traditional thermodynamics is formulated on the basis of equilibrium states in which the macroscopic properties of the system no longer change, and the free energy is at a minimum. A living thing, however, is better described as existing in a nonequilibrium steady state — one requiring a constant flow of energy and mass through the system. The challenge is to relate these seemingly different states, extending the theory of thermodynamics to make predictions about small systems and individual molecules.

Using optical tweezers, Bustamante, Liphardt, and their colleagues performed pioneering experiments on the mechanical unfolding of RNA molecules, which established that a mathematical relation known as the Jarzynski Equality is applicable in exploring how energy states govern the ways large biological molecules fold. More recently Liphardt, Bustamante, and others, including equality author Chris Jarzynski from Los Alamos, have measured the statistical properties of microspheres (beads) driven through water by optical tweezers.

A bead fluctuating in an optical trap balances the frictional force of the water through which it moves and the optical forces holding it in the trap; at different speeds, these fluctuations constitute different nonequilibrium steady states. The results of the study showed that the Jarzynski Equality can be used to recover thermodynamic information about nanoscale systems previously thought inaccessible, effectively extending thermodynamics into the realm of living things — including biobots.

"We are developing theoretical tools to understand the perturbation of small systems; we're building the hybrid instruments we need to assemble and evaluate nanoscale structures. Next is building an actual device," says Liphardt. "Only by doing it the hard way can we show we're on the right track."

The Wright brothers' first airplane didn't carry freight or passengers, and the very first biobot may not do much more than prove it can move and respond to its surroundings. Nevertheless, says Liphardt, "Our biobot program is helping to lay the foundations of a future science of molecular architecture. We're all going to be surprised at the remarkable developments just around the corner."

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