(152a) Optimizing Biological Circuits: Integrating Rational Design with Directed Evolution

The field of synthetic gene circuits is predicated on
this tenet: the ability to design and implement a desired circuit
functionality yields insight into nature's
underlying design
principles [9,11].
Consequently, this understanding should greatly impact biomedical
research as we learn how to `program' cellular behavior. Studies have
demonstrated the feasibility of building gene circuits that lead to,
for example, bi-stable toggle switches [8],
oscillatory responses [4],
and metabolic controllers [5].
While these initial studies of intracellular processes highlight the
potential of forward engineering biological entities, the dream,
however, far outstrips the reality: while the genome book is hard to
read, it is even more difficult to
(re)write. Synthetic gene circuits magnify this difficulty because
most circuits attempt to construct novel combinations of
regulatory elements coming from diverse organisms.

We propose to overcome this problem by integrating rational
design with directed evolution. We advocate using rational design
(i.e., mathematical modeling) to identify mutational targets for
non-functional circuits. Directed evolution can then be used to first
systematically perturb the mutational target to generate components
with a wide range of functionality, at which point circuit behavior can
be evaluated
for each component. This procedure builds upon several recent works.
Feng et al. also propose using models and random mutagenesis to
respectively identify and perturb mutational targets,
but suggest screening for functional behavior in the nominal
circuit [6]. In contrast,
we propose to mutate and characterize these targets with circuits of
minimal complexity (i.e., not in the nominal circuit). Characterization of
the nominal circuit via systematic perturbation of a single parameter
is conceptually similar to the strategy of Alper et
al. [1], with the
distinction that the targets are not restricted to constitutive
promoters. The benefits of this approach are three-fold. First,
performing directed evolution on a circuit of minimal complexity
ensures that the resulting library covers a wide range of parameter
values and facilitates trouble-shooting. Second, the generated
library consists of interchangeable components that (1) enable
``plug and play'' alteration of the circuit by simply exchanging one
component for another and (2) can readily be
applied to other generic circuits. Finally, examining
the circuit behavior over a library of components with widely-varying
parameter values facilitates model validation and refinement.

We apply this strategy to examine the Lux quorum-sensing module.
Natively found in the marine bacterium Vibrio fischeri, a
facultative symbiont of luminescent fish or squid, this
module regulates gene expression as a function of the population
density [7]. The
key element of this system is a regulatory cassette consisting of
genes encoding LuxI and LuxR. LuxI is an acyl-homoserine lactone (AHL)
synthase; LuxR is a
transcriptional regulator activated by the AHL. The AHL signal
molecule is produced inside the cell, but can freely diffuse
across the cell membrane into the environment. Therefore, the AHL
concentration is low at low cell density. As the cell density
increases, the signal accumulates in the environment and inside the
cell. AHL can bind and activate LuxR, activating downstream genes only
when the concentration exceeds a threshold due to a positive-feedback
mechanism. To better understand this regulatory module, we have
constructed design configurations in which different combinations of
LuxI and LuxR are controlled through positive feedback. As
suggested by a mathematical model, we have probed the possible ranges
of system behavior for each of
these configurations by employing DNA-binding affinity mutants of LuxR.
The altered affinities of these mutants are characterized in a circuit
of minimal complexity using a
fluorescence-based assay [3].
The results demonstrate that simple recombination events
combined with point mutations can readily alter the steady-state
characteristics of this module. These results provide a better
quantitative understanding of how bacteria regulate cell-cell
communication and shed light upon how the natural quorum-sensing
configuration might have evolved. We expect these findings to provide
insights into controlling pathogens that regulate virulence factors
via quorum sensing [10] and
facilitate re-engineering of quorum-sensing components for tasks such as
cancer therapy [2].

References

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2

J. C. Anderson, E. J. Clarke, A. P. Arkin, and C. A. Voigt.

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3

C. H. Collins, F. H. Arnold, and J. R. Leadbetter.

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J. Hasty, D. McMillen, and J. J. Collins.

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