A New Conditional Transcription Factor for Use in Toxoplasma Gondii.

Sharifpour, MF; Khadiv, S; Uboldi, AD; Jayawickrama, AA; Manning, S; Whittall, J; Bottema, CDK; Ellis, JT; Howe, DK; Wilson, DW; Hemmatzadeh, F; Tonkin, CJ; McAllister, MM

A New Conditional Transcription Factor for Use in Toxoplasma Gondii.

Sharifpour, MF Khadiv, S Uboldi, AD Jayawickrama, AA Manning, S Whittall, J Bottema, CDK Ellis, JT Howe, DK Wilson, DW Hemmatzadeh, F Tonkin, CJ McAllister, MM

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Publisher:: Wiley
Publication Type:: Journal Article
Citation:: Biotechnol J, 2026, 21, (5), pp. e70240
Issue Date:: 2026-05

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Field	Value	Language
dc.contributor.author	Sharifpour, MF
dc.contributor.author	Khadiv, S
dc.contributor.author	Uboldi, AD
dc.contributor.author	Jayawickrama, AA
dc.contributor.author	Manning, S
dc.contributor.author	Whittall, J
dc.contributor.author	Bottema, CDK
dc.contributor.author	Ellis, JT
dc.contributor.author	Howe, DK
dc.contributor.author	Wilson, DW
dc.contributor.author	Hemmatzadeh, F
dc.contributor.author	Tonkin, CJ
dc.contributor.author	McAllister, MM
dc.date.accessioned	2026-10-09T02:20:05Z
dc.date.available	2026-04-29
dc.date.available	2026-10-09T02:20:05Z
dc.date.issued	2026-05
dc.identifier.citation	Biotechnol J, 2026, 21, (5), pp. e70240
dc.identifier.issn	1860-6768
dc.identifier.issn	1860-7314
dc.identifier.uri	http://hdl.handle.net/10453/196745
dc.description.abstract	Toxoplasma gondii is an intracellular eukaryotic parasite in the phylum Apicomplexa. Reversible, conditional control of gene transcription was achieved previously in this organism using a tet-off system that was controlled by tetracycline. Although revolutionary for the field at the time, that system in Toxoplasma yielded modest signal-to-noise ratios (SNR) and proved to be leaky in the "off" state (i.e., it suffered from high background expression). Here we report the development of a new reversible and robust Conditional Transcription Factor (CTF) that is controlled by rapamycin. Although we originally intended to design a dimerizable drug-on transcription control system, the CTF unexpectedly functions in a drug-off fashion. In stably-transfected tachyzoites, CTF regulates a fluorescent reporter gene in Toxoplasma to achieve almost no background expression in the presence of rapamycin (i.e. near-zero leakiness), while in the absence of rapamycin 84% of tachyzoites express EYFP in comparison with 87% of constitutively-expressed positive controls, with 59% mean fluorescence intensity relative to positive controls. Conditional regulation of the median fluorescence intensity of the reporter gene reached an impressive SNR averaging 1489, approximating an "on/off" switch. The modular design of the CTF is expected to facilitate application to varied genes, incremental modifications, and adaptation for use in other apicomplexan organisms.
dc.format	Print
dc.language	eng
dc.publisher	Wiley
dc.relation.ispartof	Biotechnol J
dc.relation.isbasedon	10.1002/biot.70240
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	1002 Environmental Biotechnology, 1003 Industrial Biotechnology, 1004 Medical Biotechnology
dc.subject.classification	Biotechnology
dc.subject.classification	3101 Biochemistry and cell biology
dc.subject.classification	3106 Industrial biotechnology
dc.subject.mesh	Transcription Factors
dc.subject.mesh	Toxoplasma
dc.subject.mesh	Sirolimus
dc.subject.mesh	Gene Expression Regulation
dc.subject.mesh	Genes, Reporter
dc.subject.mesh	Signal-To-Noise Ratio
dc.subject.mesh	Transcription, Genetic
dc.subject.mesh	Genetic Vectors
dc.subject.mesh	Luminescent Proteins
dc.subject.mesh	Toxoplasma
dc.subject.mesh	Sirolimus
dc.subject.mesh	Luminescent Proteins
dc.subject.mesh	Transcription Factors
dc.subject.mesh	Transcription, Genetic
dc.subject.mesh	Gene Expression Regulation
dc.subject.mesh	Genes, Reporter
dc.subject.mesh	Genetic Vectors
dc.subject.mesh	Signal-To-Noise Ratio
dc.subject.mesh	Transcription Factors
dc.subject.mesh	Toxoplasma
dc.subject.mesh	Sirolimus
dc.subject.mesh	Gene Expression Regulation
dc.subject.mesh	Genes, Reporter
dc.subject.mesh	Signal-To-Noise Ratio
dc.subject.mesh	Transcription, Genetic
dc.subject.mesh	Genetic Vectors
dc.subject.mesh	Luminescent Proteins
dc.title	A New Conditional Transcription Factor for Use in Toxoplasma Gondii.
dc.type	Journal Article
utslib.citation.volume	21
utslib.location.activity	Germany
utslib.for	1002 Environmental Biotechnology
utslib.for	1003 Industrial Biotechnology
utslib.for	1004 Medical Biotechnology
pubs.organisational-group	University of Technology Sydney
pubs.organisational-group	University of Technology Sydney/Faculty of Science
pubs.organisational-group	University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	open_access	*
dc.rights.license	This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0). To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-nd/4.0/
dc.date.updated	2026-10-09T02:20:04Z
pubs.issue	5
pubs.publication-status	Published
pubs.volume	21
utslib.citation.issue	5

Abstract:

Toxoplasma gondii is an intracellular eukaryotic parasite in the phylum Apicomplexa. Reversible, conditional control of gene transcription was achieved previously in this organism using a tet-off system that was controlled by tetracycline. Although revolutionary for the field at the time, that system in Toxoplasma yielded modest signal-to-noise ratios (SNR) and proved to be leaky in the "off" state (i.e., it suffered from high background expression). Here we report the development of a new reversible and robust Conditional Transcription Factor (CTF) that is controlled by rapamycin. Although we originally intended to design a dimerizable drug-on transcription control system, the CTF unexpectedly functions in a drug-off fashion. In stably-transfected tachyzoites, CTF regulates a fluorescent reporter gene in Toxoplasma to achieve almost no background expression in the presence of rapamycin (i.e. near-zero leakiness), while in the absence of rapamycin 84% of tachyzoites express EYFP in comparison with 87% of constitutively-expressed positive controls, with 59% mean fluorescence intensity relative to positive controls. Conditional regulation of the median fluorescence intensity of the reporter gene reached an impressive SNR averaging 1489, approximating an "on/off" switch. The modular design of the CTF is expected to facilitate application to varied genes, incremental modifications, and adaptation for use in other apicomplexan organisms.

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/196745