Regulation of Bacterial Gene Expression by Protease-Alleviated
Spatial Sequestration (PASS)
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Abstract
In
natural microbial systems, conditional spatial sequestration of transcription
factors enables cells to respond rapidly to changes in their environment
or intracellular state by releasing presynthesized regulatory proteins.
Although such a mechanism may be useful for engineering synthetic
biology technologies ranging from cell-based biosensors to biosynthetic
platforms, to date it remains unknown how or whether such conditional
spatial sequestration may be engineered. In particular, based upon
seemingly contradictory reports in the literature, it is not clear
whether subcellular spatial localization of a transcription factor
within the cytoplasm is sufficient to preclude regulation of cognate
promoters on plasmid-borne or chromosomal loci. Here, we describe
a modular, orthogonal platform for investigating and implementing
this mechanism using protease-alleviated spatial sequestration (PASS).
In this system, expression of an exogenous protease mediates the proteolytic
release of engineered transcriptional regulators from the inner face
of the <i>Escherichia coli</i> cytoplasmic membrane. We demonstrate that PASS mediates robust,
conditional regulation of either transcriptional repression, <i>via</i> tetR, or transcriptional activation, by the 位
phage CI protein. This work provides new insights into a biologically
important facet of microbial gene expression and establishes a new
strategy for engineering conditional transcriptional regulation for
the microbial synthetic biology toolbox