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Volume 15
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10.3390/toxins15080508
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Article

A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli

by
Jessie Vandierendonck
†,
Yana Girardin
†,
Pieter De Bruyn
,
Henri De Greve
* and
Remy Loris
*
Center for Structural Biology, Vlaams Instituut voor Biotechnologie and Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Toxins 2023, 15(8), 508; https://doi.org/10.3390/toxins15080508
Submission received: 5 April 2023 / Revised: 17 July 2023 / Accepted: 8 August 2023 / Published: 18 August 2023
(This article belongs to the Section Bacterial Toxins)
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Abstract

Molecular cloning and controlled expression remain challenging when the target gene encodes a protein that is toxic to the host. We developed a set of multi-layer control systems to enable cloning of genes encoding proteins known to be highly toxic in Escherichia coli and other bacteria. The different multi-layer control systems combine a promoter–operator system on a transcriptional level with a riboswitch for translational control. Additionally, replicational control is ensured by using a strain that reduces the plasmid copy number. The use of weaker promoters (such as PBAD or PfdeA) in combination with the effective theophylline riboswitch is essential for cloning genes that encode notoriously toxic proteins that directly target translation and transcription. Controlled overexpression is possible, allowing the system to be used for evaluating in vivo effects of the toxin. Systems with a stronger promoter can be used for successful overexpression and purification of the desired protein but are limited to toxins that are more moderate and do not interfere with their own production.
Keywords: toxins; cloning; riboswitch; transcriptional control; translational control; replicational control toxins; cloning; riboswitch; transcriptional control; translational control; replicational control

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MDPI and ACS Style

Vandierendonck, J.; Girardin, Y.; De Bruyn, P.; De Greve, H.; Loris, R. A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli. Toxins 2023, 15, 508. https://doi.org/10.3390/toxins15080508

AMA Style

Vandierendonck J, Girardin Y, De Bruyn P, De Greve H, Loris R. A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli. Toxins. 2023; 15(8):508. https://doi.org/10.3390/toxins15080508

Chicago/Turabian Style

Vandierendonck, Jessie, Yana Girardin, Pieter De Bruyn, Henri De Greve, and Remy Loris. 2023. "A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli" Toxins 15, no. 8: 508. https://doi.org/10.3390/toxins15080508

APA Style

Vandierendonck, J., Girardin, Y., De Bruyn, P., De Greve, H., & Loris, R. (2023). A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli. Toxins, 15(8), 508. https://doi.org/10.3390/toxins15080508

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