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(102c) Engineering NAND and NOR Logic Gates Using Transcriptional Interference

O'Connor, N., University of Colorado Boulder
Bordoy, A. E., University of Colorado Boulder
Chatterjee, A., University of Colorado Boulder
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Nolan O'Connor Normal Nolan O'Connor 2 131 2019-04-12T21:45:00Z 2019-04-12T21:45:00Z 1 252 1438 11 3 1687 16.00

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" times new roman>Antisense transcription is widespread in all kingdoms
of life and has been shown to regulate important cellular decisions. Regulation
may occur both through mechanisms of transcriptional interference (TI)—
including head-on collision of converging RNA Polymerases (RNAPs)— and
interactions between sense and antisense transcripts. Mathematical
modeling and experiments have characterized several naturally occurring TI
systems, but TI is currently under-used in synthetic biology and the design
rules for constructing TI-based genetic devices are not yet well-defined. Here,
we present a novel TI system in which Rho termination of the interfering RNAP
is manipulated in order to tune the extent of TI repression. We
demonstrate that the interfering RNAP’s co-translation with a ribosome is
essential for strong TI because it prevents Rho termination of RNA Polymerases
in untranslated regions. We also show that TI strength can be tuned by
modulating the length of the open reading frame in a convergent gene construct,
tuning the ribosome binding site strength of the interfering gene, and
inhibiting Rho termination of the interfering RNA Polymerase. We then apply
these design rules to build two-input, minimal NAND and NOR transcriptional
logic gates that achieve >40- fold gene repression. The behaviors of these
gates are then validated and predicted through a mathematical modeling approach
that merges stochastic modeling of RNAP collisions with transfer functions describing
operator and promoter occupancy. These results expand the potential for TI as a
novel tool for synthetic biology and offer insights into an important but
relatively unexplored gene regulatory mechanism.


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