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ACS Synth Biol. 2014 Sep 19;3(9):666-75. doi: 10.1021/sb500084f. Epub 2014 Jul 28.

Expanding the logic of bacterial promoters using engineered overlapping operators for global regulators.

Author information

1
FFCLRP and ‡FMRP, University of São Paulo , Ribeirão Preto, SP Brazil.

Abstract

The understanding of how the architecture of cis-regulatory elements at bacterial promoters determines their final output is of central interest in modern biology. In this work, we attempt to gain insight into this process by analyzing complex promoter architectures in the model organism Escherichia coli. By focusing on the relationship between different TFs at the genomic scale in terms of their binding site arrangement and their effect on the target promoters, we found no strong constraint limiting the combinatorial assembly of TF pairs in E. coli. More strikingly, overlapping binding sites were found equally associated with both equivalent (both TFs have the same effect on the promoter) and opposite (one TF activates while the other repress the promoter) effects on gene expression. With this information on hand, we set an in silico approach to design overlapping sites for three global regulators (GRs) of E. coli, specifically CRP, Fis, and IHF. Using random sequence assembly and an evolutionary algorithm, we were able to identify potential overlapping operators for all TF pairs. In order to validate our prediction, we constructed two lac promoter variants containing overlapping sites for CRP and IHF designed in silico. By assaying the synthetic promoters using a GFP reporter system, we demonstrated that these variants were functional and activated by CRP and IHF in vivo. Taken together, presented results add new information on the mechanisms of signal integration in bacterial promoters and provide new strategies for the engineering of synthetic regulatory circuits in bacteria.

KEYWORDS:

cis-regulatory elements; regulatory networks; synthetic biology

PMID:
25036188
DOI:
10.1021/sb500084f
[Indexed for MEDLINE]

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