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Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor

(2022) GENE. 809.
Author
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Abstract
Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators. This work aims to design and explore the use of an archaeal TetR family regulator, FadRSa from Sulfolobus acidocaldarius, in a bacterial system, namely Escherichia coli. This is a challenging objective given the fundamental difference between the bacterial and archaeal transcription machinery and the lack of a native TetR-like FadR regulatory system in E. coli. The synthetic a70-dependent bacterial promoter proD was used as a starting point to design hybrid bacterial/archaeal promoter/operator regions, in combination with the mKate2 fluorescent reporter enabling a readout. Four variations of proD containing FadRSa binding sites were constructed and characterized. While expressional activity of the modified promoter proD was found to be severely diminished for two of the constructs, constructs in which the binding site was introduced adjacent to the -35 promoter element still displayed sufficient basal transcriptional activity and showed up to 7fold repression upon expression of FadRSa. Addition of acyl-CoA has been shown to disrupt FadRSa binding to the DNA in vitro. However, extracellular concentrations of up to 2 mM dodecanoate, subsequently converted to acylCoA by the cell, did not have a significant effect on repression in the bacterial system. This work demonstrates that archaeal transcription regulators can be used to generate actuator elements for use in E. coli, although the lack of ligand response underscores the challenge of maintaining biological function when transferring parts to a phylogenetically divergent host.
Keywords
Genetics, General Medicine, Synthetic biology, Sulfolobus, acyl-CoA, Hybrid promoter, operator, FadR, GENE-EXPRESSION, DESIGN, PATHWAYS, PROTEIN

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MLA
Sybers, David, et al. “Engineering Transcriptional Regulation in Escherichia Coli Using an Archaeal TetR-Family Transcription Factor.” GENE, vol. 809, 2022, doi:10.1016/j.gene.2021.146010.
APA
Sybers, D., Joka Bernauw, A., El Masri, D., Ramadan Maklad, H., Charlier, D., De Mey, M., … Peeters, E. (2022). Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor. GENE, 809. https://doi.org/10.1016/j.gene.2021.146010
Chicago author-date
Sybers, David, Amber Joka Bernauw, Diala El Masri, Hassan Ramadan Maklad, Daniel Charlier, Marjan De Mey, Indra Bervoets, and Eveline Peeters. 2022. “Engineering Transcriptional Regulation in Escherichia Coli Using an Archaeal TetR-Family Transcription Factor.” GENE 809. https://doi.org/10.1016/j.gene.2021.146010.
Chicago author-date (all authors)
Sybers, David, Amber Joka Bernauw, Diala El Masri, Hassan Ramadan Maklad, Daniel Charlier, Marjan De Mey, Indra Bervoets, and Eveline Peeters. 2022. “Engineering Transcriptional Regulation in Escherichia Coli Using an Archaeal TetR-Family Transcription Factor.” GENE 809. doi:10.1016/j.gene.2021.146010.
Vancouver
1.
Sybers D, Joka Bernauw A, El Masri D, Ramadan Maklad H, Charlier D, De Mey M, et al. Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor. GENE. 2022;809.
IEEE
[1]
D. Sybers et al., “Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor,” GENE, vol. 809, 2022.
@article{8726519,
  abstract     = {{Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators. This work aims to design and explore the use of an archaeal TetR family regulator, FadRSa from Sulfolobus acidocaldarius, in a bacterial system, namely Escherichia coli. This is a challenging objective given the fundamental difference between the bacterial and archaeal transcription machinery and the lack of a native TetR-like FadR regulatory system in E. coli. The synthetic a70-dependent bacterial promoter proD was used as a starting point to design hybrid bacterial/archaeal promoter/operator regions, in combination with the mKate2 fluorescent reporter enabling a readout. Four variations of proD containing FadRSa binding sites were constructed and characterized. While expressional activity of the modified promoter proD was found to be severely diminished for two of the constructs, constructs in which the binding site was introduced adjacent to the -35 promoter element still displayed sufficient basal transcriptional activity and showed up to 7fold repression upon expression of FadRSa. Addition of acyl-CoA has been shown to disrupt FadRSa binding to the DNA in vitro. However, extracellular concentrations of up to 2 mM dodecanoate, subsequently converted to acylCoA by the cell, did not have a significant effect on repression in the bacterial system. This work demonstrates that archaeal transcription regulators can be used to generate actuator elements for use in E. coli, although the lack of ligand response underscores the challenge of maintaining biological function when transferring parts to a phylogenetically divergent host.}},
  articleno    = {{146010}},
  author       = {{Sybers, David and Joka Bernauw, Amber and El Masri, Diala and Ramadan Maklad, Hassan and Charlier, Daniel and De Mey, Marjan and Bervoets, Indra and Peeters, Eveline}},
  issn         = {{0378-1119}},
  journal      = {{GENE}},
  keywords     = {{Genetics,General Medicine,Synthetic biology,Sulfolobus,acyl-CoA,Hybrid promoter,operator,FadR,GENE-EXPRESSION,DESIGN,PATHWAYS,PROTEIN}},
  language     = {{eng}},
  pages        = {{9}},
  title        = {{Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor}},
  url          = {{http://doi.org/10.1016/j.gene.2021.146010}},
  volume       = {{809}},
  year         = {{2022}},
}

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