The computer assisted design (CAD) tools that made
it possible to fabricate integrated circuits with millions of transistors may
soon be coming to the biological sciences. Researchers at the U.S. Department
of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have developed CAD-type
models and simulations for RNA molecules that make it possible to engineer
biological components or “RNA devices” for controlling genetic expression in
microbes. This holds enormous potential for microbial-based sustainable production
of advanced biofuels, biodegradable plastics, therapeutic drugs, and a host of
other goods now derived from petrochemicals.
“Because biological systems exhibit functional
complexity at multiple scales, a big question has been whether effective design
tools can be created to increase the sizes and complexities of the microbial
systems we engineer to meet specific needs,” says Jay Keasling, director of
JBEI and a world authority on synthetic biology and metabolic engineering. “Our
work establishes a foundation for developing CAD platforms to engineer complex
RNA-based control systems that can process cellular information and program the
expression of very large numbers of genes. Perhaps even more importantly, we
have provided a framework for studying RNA functions and demonstrated the
potential of using biochemical and biophysical modeling to develop rigorous
design-driven engineering strategies for biology.”
Keasling, who also holds appointments with the
Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of
California (UC) Berkley,
is the corresponding author of a paper in Science that describes this
work. The paper is titled “Model-driven engineering of RNA devices to
quantitatively-program gene expression.” Other co-authors are James Carothers,
Jonathan Goler, and Darmawi Juminaga.
Synthetic biology is an emerging scientific field
in which novel biological devices, such as molecules, genetic circuits or
cells, are designed and constructed, or existing biological systems, such as
microbes, are re-designed and engineered. A major goal is to produce valuable
chemical products from simple, inexpensive and renewable starting materials in
a sustainable manner. As with other engineering disciplines, CAD tools for
simulating and designing global functions based upon local component behaviors
are essential for constructing complex biological devices and systems. However,
until this work, CAD-type models and simulation tools for biology have been
very limited.
“Identifying the relevant design parameters and
defining the domains over which expected component behaviors are exerted have
been key steps in the development of CAD tools for other engineering
disciplines,” says Carothers, a bioengineer and lead author of the Science
paper who is a member of Keasling’s research groups with both JBEI and the
California Institute for Quantitative Biosciences. “We’ve applied generalizable
engineering strategies for managing functional complexity to develop CAD-type
simulation and modeling tools for designing RNA-based genetic control systems.
Ultimately we’d like to develop CAD platforms for synthetic biology that rival
the tools found in more established engineering disciplines, and we see this
work as an important technical and conceptual step in that direction.”
Keasling, Carothers, and their co-authors focused
their design-driven approach on RNA sequences that can fold into complicated
three dimensional shapes, called ribozymes and aptazymes. Like proteins,
ribozymes and aptazymes can bind metabolites, catalyze reactions, and act to
control gene expression in bacteria, yeast, and mammalian cells. Using
mechanistic models of biochemical function and kinetic biophysical simulations
of RNA folding, ribozyme and aptazyme devices with quantitatively predictable
functions were assembled from components that were characterized in vitro,
in vivo, and in silico. The models and design strategy were
then verified by constructing 28 genetic expression devices for the Escherichia
coli bacterium. When tested, these devices showed excellent agreement—94%
correlation—between predicted and measured gene expression levels.
“We needed to formulate models that would be
sophisticated enough to capture the details required for simulating system
functions, but simple enough to be framed in terms of measurable and tunable
component characteristics or design variables,” Carothers says. “We think of
design variables as the parts of the system that can be predictably modified,
in the same way that a chemical engineer might tune the operation of a chemical
plant by turning knobs that control fluid flow through valves. In our case,
knob-turns are represented by specific kinetic terms for RNA folding and
ribozyme catalysis, and our models are needed to tell us how a combination of
these knob-turns will affect overall system function.”
JBEI researchers are now using their RNA CAD-type
models and simulations as well as the ribozyme and aptazyme devices they
constructed to help them engineer metabolic pathways that will increase
microbial fuel production. JBEI is one of three DOE Bioenergy Research Centers
established by DOE’s Office of Science to advance the technology for the
commercial production of clean, green, and renewable biofuels. A key to JBEI’s
success will be the engineering of microbes that can digest lignocellulosic
biomass and synthesize from the sugars transportation fuels that can replace
gasoline, diesel, and jet fuels in today’s engines.
“In addition to advanced biofuels, we’re also
looking into engineering microbes to produce chemicals from renewable
feedstocks that are difficult to produce cheaply and in high yield using
traditional organic chemistry technology,” Carothers says.
While the RNA models and simulations developed at
JBEI to date fall short of being a full-fledged RNA CAD platform, Keasling,
Carothers, and their coauthors are moving towards that goal.
“We are also actively trying to make our models and
simulations more accessible to researchers who may not want to become RNA control
system experts but would nonetheless like to use our approach and RNA devices
in their own work,” Carothers says.
While the work at JBEI focused on E. coli and
the microbial production of advanced biofuels, the authors of the Science
paper believe that their concepts could also be used for programming function
into mammalian systems and cells.
“We recently initiated a research project to
investigate how we can use our approach to engineer RNA-based genetic control
systems that will increase the safety and efficacy of regenerative medicine
therapies that use cultured stem cells to treat diseases such as diabetes and
Parkinson’s,” Carothers says.