(563f) An in vitro Transcriptional Regulatory Network for Modular Control of Synthetic Signals

An
in vitro transcriptional regulatory network for modular control of
synthetic signals

Samuel W. Schaffter^a
and Rebecca Schulman^{a, c}

^aDepartment of Chemical and Biomolecular
Engineering, ^cDepartment of Computer Science – Johns Hopkins
University

In biology, cells and organisms
are capable of undergoing complex transformations in response to environmental
stimuli. These transformations are orchestrated by genetic regulatory networks
(GRNs) which are collections of genes that interact with each other to control
cellular function. Environmental signals are translated into signaling cascades
through these integrated networks to promote changes in gene expression that
can regulate downstream signals expressed within the cell. These downstream
signals ultimately change cellular function. A route to mimic this complicated
regulation in engineered systems is to develop an in vitro architecture that
enables control of synthetic signals in a manner similar to GRNs. A possible
architecture for instituting regulatory network-like control on synthetic
signals is through in vitro transcriptional circuits. These circuits
consist of short synthetic genelets that are transcribed by viral RNA
polymerases and utilize only nucleic acids to regulate genelet expression, making
them highly programmable and simpler than translation based in vitro
circuits. While common GRN motifs have been constructed using genelets, little
work has been done to build responsive networks, limiting their use as regulatory
networks. Furthermore, current genelet networks have
been limited to a small set of orthogonally controlled genelets due to non-specific
hybridization reactions between circuit components (crosstalk) that arise as
more components are added. Thus, it is difficult to build large layered genelet
based networks as seen in cellular GRNs.

Here
we construct an in vitro transcriptional regulatory network with
genelets that modularly controls the switchable expression of downstream
synthetic RNA signals. We start by developing a responsive bistable motif which
can be switched back and forth between its two stable steady states with
specifically designed RNAs. We next develop a new genelet design that allows
orthogonally controlled genelets to be added to an existing genelet network
without introducing component crosstalk. We utilize this genelet design to connect
our bistable network to upstream network nodes and demonstrate that genelet
networks can be reliably scaled up. We then demonstrate that this expanded
network can modularly switch between the production of distinct downstream RNA
signals. Finally, we integrate a signal fan-out feedforward loop into the
network to rapidly reset the network when the signal to switch states is
presented. This genelet regulatory network serves as a highly programmable in
vitro analogue to cellular GRNs and could be utilized to drive chemical
processes in engineered systems. The RNA signals produced by our regulatory
network could be utilized to drive state changes in more complex downstream
processes, such as nucleic acid driven nanostructure organization. Additionally,
the network could be utilized to produce switchable chemical gradients or
patterns. Through the use of aptamers, the RNA signals could be expanded to control
the activity of small molecules or proteins. Furthermore, the modular design
and scalability of this regulatory network allows it to be integrated into
larger networks to enable more complex regulatory behavior.