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. 2018 Jun 2;102(15):6357–6372. doi: 10.1007/s00253-018-9115-1

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© The Author(s) 2018

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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Fig. 1 — Tet-Off expression system. a Basic version of the Tet-Off expression system in eukaryotes. The fusion of TetR with VP16 leads to the generation of the tetracyclin-dependent transactivator tTA. When tetracycline or its analog doxycycline is added, tTA is unable to specifically bind to tetO due to a conformational change. As a result, transcription of the gene of interest, which is controlled by Pmin, is disrupted. Thus, transcriptional activation takes place in the absence of tetracycline or doxycycline. Addition of the either drug leads to transcriptional shutdown. Dox, doxycycline; VP16, transcriptional activator domain from herpes simplex virus; tc, tetracycline; TetR, tetracycline-dependent Tet repressor; tetO; tetracycline operator sequence; tTA, tetracyclin-dependent transactivator; TtrpC, trpC terminator from Aspergillus nidulans; Pmin, minimal promoter. b Optimized module of the Tet-Off expression system for A. niger. The transactivator tTA2^S, optimized for mammals, was used downstream of PgpdA promoter. The promoter can be selected individually depending on the model organism (PgeneX). Seven copies of tetO sequence were inserted (tetO7) upstream of a minimal promoter in order to increase tTA2^S binding. In the absence of tetracycline or its analog doxycycline tTA2^S is able to specifically bind tetO7. Thus, the transcriptional induction of the gene of interest, which is controlled by Pmin takes place. Addition of drug leads to transcriptional shutdown. For further details, see text. Abbreviations: Dox, doxycycline; VP16, transcriptional activator domain from herpes simplex virus tc, tetracycline; TetR, tetracycline-dependent Tet repressor; tetO, tetracycline operator sequence; tetO7, seven copies of tetracycline operator sequence; tTA, tetracyclin-dependent transactivator; tTA2^S, mammalian tetracyclin-dependent transactivator; TcrgA, crgA terminator from Aspergillus fumigatus; TtrpC, trpC terminator from Aspergillus nidulans; Pmin, minimal promoter (adopted from Brockamp et al. 2002, Wanka et al. 2016)