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“If you don’t like the news, go out and make some of your own.” . . . Wes “Scoop” Nisker.
Taking
a page from the book of San Francisco radio legend Scoop Nisker,
biologists who find themselves dissatisfied with the microbes nature has
provided are going out and making some of their own. Members of the
fast-growing “synthetic biology” research community are designing and
constructing novel organisms and biologically-inspired systems – or
redesigning existing organisms and systems – to solve problems that
natural systems cannot. The range of potential applications for
synthetic biological systems runs broad and deep, and includes such
profoundly important ventures as the microbial-based production of
advanced biofuels and inexpensive versions of critical therapeutic
drugs.
Synthetic
biology, however, is still a relatively new scientific field plagued
with the trial and error inefficiencies that hamper most technologies in
their early stages of development. To help address these problems,
synthetic biologists aim to create biological circuits that can be used
for the safer and more efficient construction of increasingly complex
functions in microorganisms. A central component of such circuits is
RNA, the multipurpose workhorse molecule of biology.
“A
widespread natural ability to sense small molecules and regulate genes
has made the RNA molecule an important tool for synthetic biology in
applications as diverse as environmental sensing and metabolic
engineering,” says Adam Arkin, a computational biologist with the U.S.
Department of Energy (DOE)’s Lawrence Berkeley National Laboratory
(Berkeley Lab), where he serves as director of the Physical Biosciences
Division. Arkin is also a professor at the University of California (UC)
Berkeley where he directs the Synthetic Biology Institute, a
partnership between UC Berkeley and Berkeley Lab.
From left Julius Lucks, Lei “Stanley” Qi and Adam Arkin created an RNA-based regulatory system that can independently control the transcription activities of multiple targets in a single cell. (Photo by Roy Kaltschmidt, Berkeley Lab Public Affairs) |
In
his multiple capacities, Arkin is leading a major effort to use RNA
molecules for the engineering of programmable genetic networks. In
recent years, scientists have learned that the behavior of cells is
often governed by multiple different genes working together in networked
teams that are regulated through RNA-based mechanisms. Synthetic
biologists have been using RNA regulatory mechanisms to program genetic
networks in cells to achieve specific results. However, to date these
programming efforts have required proteins to propagate RNA regulatory
signals. This can pose problems because one of the primary goals of
synthetic biology is to create families of standard genetic parts that
can be combined to create biological circuits with behaviors that are to
some extent predictable. Proteins can be difficult to design and
predict. They also add a layer of complexity to biological circuits that
can delay and slow the dynamics of the circuit’s responses.
“We’re now able to eliminate the protein requirement and directly propagate regulatory signals as RNA molecules,” Arkin says.
Working
with their own variations of RNA transcription attenuators – nucleotide
sequences that under a specific set of conditions will stop the RNA
transcription process – Arkin and his colleagues engineered a system in
which these independent attenuators can be configured to sense RNA input
and synthesize RNA output signals. These variant RNA attenuators can
also be configured to regulate multiple genes in the same cell and –
through the controlled expression of these genes – perform logic
operations.
The circuitry of biological cells is not unlike electronic circuitry as shown in this schematic of E. coli metabolic reactions. Synthetic biologists want to create standard genetic parts for the production of their own biological circuits. |
“We
have demonstrated the ability to construct with minimal changes
orthogonal variants of natural RNA transcription attenuators that
function more or less homogeneously in a single regulatory system, and
we have shown that the composition of this system is predictable,” Arkin
says. “This is the first time that the three regulatory features of our
system, which are all properties featured in a semiconductor
transistor, have been captured in a single biological molecule.”
A
paper describing this breakthrough appears in the Proceedings of the
National Academy of Science (PNAS) under the title “Versatile
RNA-sensing transcriptional regulators for engineering genetic
networks”. Co-authoring the paper with Arkin were first authors Julius
Lucks and Lei “Stanley” Qi, plus Vivek Mutalik and Denise Wang.
The
success of Arkin and his colleagues was based on their making use of an
element in the bacterial plasmid (Staphylococcus aureus)known as pT181.
The element in pT181 was an antisense RNA-mediated transcription
attenuation mechanism that controls the plasmid’s copy number. Plasmids
are molecules of DNA that serve as a standard tool of synthetic biology
for, among other applications, encoding RNA molecules. Antisense RNA
consists of non-coding nucleotide sequences that are used to regulate
genetic elements and activities, including transcription. Since the
plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism
works through RNA-to-RNA interactions, Arkin and his colleagues could
use it to create attenuator variants that would independently regulate
the transcription activity of multiple targets in the same cell – in
this case, in Escherichia coli, one of the most popular bacteria for
synthetic biology applications.
“It
is very advantageous to have independent regulatory units that control
processes such as transcription because the assembly of these units into
genetic networks follows a simple rule of composition,” Arkin says.
While
acknowledging the excellent work done on other RNA-based regulatory
mechanisms that can each perform some portion of the control functions
required for a genetic network, Arkin believes that the attenuator
variants he and his colleagues engineered provide the simplest route to
achieving all of the required control functions within a single
regulatory mechanism.
Berkeley Lab researchers are using RNA molecules to engineer genetic networks – analogous to microcircuits – into E. coli. |
“Furthermore,”
he says, “these previous efforts were fundamentally dependent on
molecular interactions through space between two or more regulatory
subunits to create a network. Our approach, which relies on the
processive transcription process, is more reliable.”
Arkin
and his colleagues say their results provide synthetic biologists with a
versatile new set of RNA-based transcriptional regulators that could
change how future genetic networks are designed and constructed. Their
engineering strategy for constructing orthogonal variants from natural
RNA system should also be applicable to other gene regulatory
mechanisms, and should add to the growing synthetic biology repertoire.
“Although
RNA has less overall functionality than proteins, its nucleic
acid-based polymer physics make mechanisms based on RNA simpler and
easier to engineer and evolve,” Arkin says. “With our RNA regulatory
system and other work in progress, we’re on our way to developing the
first complete and scalable biological design system. Ultimately, our
goal is to create a tool revolution in synthetic biology similar to the
revolution that led to the success of major integrated circuit design
and deployment.”
Much
of this research was supported by was supported by the Synthetic
Biology Engineering Research Center (SynBERC) under a grant from the
National Science Foundation.
Article by Adam Arkin and Julius Lucks titled “Synthetic Biology’s Hunt for the Genetic Transistor”
A pdf of the PNAS paper by Arkin, et. al., “Versatile RNA-sensing transcriptional regulators for engineering genetic networks”
