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A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli

Title:

A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli

Pakarian, Paknoosh and Pawelek, Peter D. (2016) A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli. BMC Biotechnology, 16 (1). p. 68. ISSN 1472-6750

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Official URL: http://dx.doi.org/10.1186/s12896-016-0298-1

Abstract

Background
In the presence of sufficient iron, the Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction.

Results
We cloned the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies.

Conclusions
The vectors pFCF1, pFCF2, and pFBH1 have been shown to use the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Refereed:Yes
Authors:Pakarian, Paknoosh and Pawelek, Peter D.
Journal or Publication:BMC Biotechnology
Date:2016
Funders:
  • Concordia Open Access Author Fund
Digital Object Identifier (DOI):10.1186/s12896-016-0298-1
Keywords:Ferric uptake regulator, Protein expression, Bidirectional promoter, Bacterial two-hybrid, CAS assay
ID Code:982267
Deposited By: DANIELLE DENNIE
Deposited On:21 Mar 2017 17:43
Last Modified:18 Jan 2018 17:54

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