Journal of Molecular Biology
Control of expression of the Tn10-encoded tetracycline resistance genes: Equilibrium and kinetic investigation of the regulatory reactions*
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2023, Science of the Total EnvironmentCitation Excerpt :After entering the bacterial cells, tetracycline molecules most likely interact with Tet repressor proteins (Argast and Beck, 1984; Levy, n.d.; Schnappinger and Hillen, 1996). Hillen et al. (1983) estimated the association constant between tetracycline and the Tet repressor proteins was >1013 L/mol. The association constant of tetracycline with EPS ranges between 103 and 107 L/mol measured in several studies (Li et al., 2021a; Li et al., 2021b; Song et al., 2014).
Thermodynamics, cooperativity and stability of the tetracycline repressor (TetR) upon tetracycline binding
2020, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :The standard free energy of dissociation of the ligand-free TetR(D) (PDB-ID 1BJZ) with respect to the buried surface in the homodimer estimated by PISA [21,22] is 30.7 kcal/mol, equivalent to KA ~1023 M−1. Gel filtration experiments of TetR(B), which shares a sequence identity with TetR(D) of 66%, revealed an estimated KA > 107 M−1 for dimer stability [26]. Melting curves measured by CD-spectrometry with increasing TetR(D) concentrations revealed no increase of Tm.
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2019, Methods in EnzymologyCitation Excerpt :The experimental data were fitted to a Hill function for a repressor with basal expression (F(x)) using the least-squares method. Noticeably, the resulting Hill coefficient (n) is approximately 2, as expected for a dimeric repressor (Hillen, Gatz, Altschmied, Schollmeier, & Meier, 1983). Cells expressing heat shock sensitive GFP, heat shock sensitive destabilized GFPODC(D12A), or the heat shock sensitive NanoDeg Inverter circuit (5 × 104 cells, 24-well plates) were treated with representative concentrations of Tc (0, 5, and 25 ng/mL) for 24 h prior to heat shock (43°C for 90 min).
Aquaporin-9 protein is the primary route of hepatocyte glycerol uptake for glycerol gluconeogenesis in mice
2011, Journal of Biological ChemistryCitation Excerpt :To identify small molecule inhibitors of AQP9, murine (m)AQP9 was introduced into CHO cells. Expression of mAQP9 was inducible by tetracycline addition to the culture medium (21, 22). Utilizing sucrose as an AQP9-impermeable osmolyte, we found that mAQP9 facilitated rapid cell shrinking in a calcein fluorescence intensity-based microplate assay (9, 23, 24).
Development of a GPR23 cell-based β-lactamase reporter assay
2010, Methods in EnzymologyCitation Excerpt :The T-REx™ System (Life Technologies) is a tetracycline (or doxycyline) inducible mammalian expression system in which the gene of interest is repressed in the absence and induced in the presence of tetracycline (Yao et al., 1998). The system makes use of regulatory elements from the E. coli Tn-10-encoded tetracycline resistance operon (Hillen and Berens, 1994; Hillen et al., 1983). There are three main components to the system which are required to achieve the inducible expression (Fig. 20.2).
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This work was supported by the Deutsche Forschungsgemeinschaft and Chemische Werke Röhm, Darmstadt.