Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria

Abstract Purpose In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. Procedures In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR. Results In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. Conclusions Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

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High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine

Plant Cell Reports ◽

10.1007/s00299-005-0009-1 ◽

2005 ◽

Vol 24 (10) ◽

pp. 619-628 ◽

Cited By ~ 5

Author(s):

Wei Tang ◽

Ronald J. Newton ◽

Thomas M. Charles

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

High Efficiency ◽

Expression System ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Chimeric Transcription Factor ◽

Inducible Gene

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Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02296-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6795-6802 ◽

Cited By ~ 40

Author(s):

Andreas Kaczmarczyk ◽

Julia A. Vorholt ◽

Anne Francez-Charlot

Keyword(s):

Gene Expression ◽

Caulobacter Crescentus ◽

Paracoccus Denitrificans ◽

Expression System ◽

Model Organisms ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Content Type ◽

Wide Range ◽

Inducible Gene

ABSTRACTTunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of thePseudomonas putidaF1cym/cmtsystem. The resulting promoter, termed PQ5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in otherAlphaproteobacteria, such as the model organismsCaulobacter crescentus,Paracoccus denitrificans, andMethylobacterium extorquens. In the noninduced state, expression from PQ5is low enough to allow gene depletion analysis, as demonstrated with the essential genephyPofSphingomonassp. strain Fr1. A set of PQ5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

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Development of an inducible gene expression system for primary murine keratinocytes

Journal of Dermatological Science ◽

10.1016/j.jdermsci.2007.09.003 ◽

2008 ◽

Vol 49 (1) ◽

pp. 73-84 ◽

Cited By ~ 6

Author(s):

Priyadharsini Nagarajan ◽

Satrajit Sinha

Keyword(s):

Gene Expression ◽

Expression System ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Inducible Gene

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Heat-Inducible Gene Expression System by Applying Alternating Magnetic Field to Magnetic Nanoparticles

ACS Synthetic Biology ◽

10.1021/sb4000838 ◽

2013 ◽

Vol 3 (5) ◽

pp. 273-279 ◽

Cited By ~ 25

Author(s):

Masaki Yamaguchi ◽

Akira Ito ◽

Akihiko Ono ◽

Yoshinori Kawabe ◽

Masamichi Kamihira

Keyword(s):

Gene Expression ◽

Magnetic Field ◽

Magnetic Nanoparticles ◽

Expression System ◽

Alternating Magnetic Field ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Inducible Gene

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A tunable anthranilate-inducible gene expression system for Pseudomonas species

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-11034-8 ◽

2020 ◽

Vol 105 (1) ◽

pp. 247-258

Author(s):

Lena Hoffmann ◽

Michael-Frederick Sugue ◽

Thomas Brüser

Keyword(s):

Gene Expression ◽

Expression System ◽

Ant System ◽

Plant Host ◽

Inducible Gene Expression ◽

Gene Expression System ◽

Expression Levels ◽

Key Points ◽

Wide Range ◽

Inducible Gene

Abstract Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads. Key points • We established an anthranilate-inducible gene expression system for pseudomonads. • This system permits tuning of gene expression in a wide range of pseudomonads. • It will be very useful for physiological and biotechnological applications.

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