Anhydrotetracycline atc

Manufactured by Takara Bio

Sourced in United States

Anhydrotetracycline (aTc) is a chemical compound used as a tool in molecular biology and biotechnology. It serves as a synthetic inducer, providing a means to regulate gene expression in genetically engineered organisms.

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Anhydrotetracycline (7 citations)

14 protocols using anhydrotetracycline atc

Topoisomerase Induction and Bacterial Growth

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E. coli strain MG1655 with chromosomally integrated YpTop1 WT (BWYTOP) or YpTop1-D117N (BW117N) bearing an N-terminal TRX tag was a gift from Prof Yuk-Ching Tse-Dinh (Florida International University, USA). Cells were cultured at 37°C in LB medium containing 25 μg/ml chloramphenicol. Top1 expression was induced in exponentially growing cultures (OD₆₀₀=0.3) by addition of arabinose (Sigma Aldrich). Cell division was monitored by measuring OD₆₀₀.
M. smegmatis (mc²155) strain was cultured in 7H9 medium with ADC supplement (HiMedia) as described (28 (link)). M. smegmatis was transformed using a MicroPulser™ Electroporator Bio-Rad), and transformed cells selected and propagated in medium containing 200 μg/ml hygromycin B (Thermo-Fisher). MtTop1 expression was induced in exponentially growing (OD₆₀₀=0.5) cultures of M. smegmatis by addition of 50 ng/ml anhydrotetracycline (ATc; Takara-Clontech) (29 (link)). Cell survival was quantified by culturing cells on LB plates containing 200 μg/ml hygromycin B for 3 days at 37°C.
M. tuberculosis H37Rv (ATCC 25618) strain was grown in 7H9 medium with ADC supplement (HiMedia) as described (28 (link)). Up to 2×10⁹ Mtb bacilli were incubated in LSB (see below) for 15 min at 65°C with occasional vortexing, washed once in water, plated, and then incubated for four weeks at 37°C.

Sinha D., Kiianitsa K., Sherman D.R, & Maizels N. (2020). Rapid, direct detection of bacterial Topoisomerase 1-DNA adducts by RADAR/ELISA. Analytical biochemistry, 608, 113827.

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Bacterial Culture Conditions and Plasmid Induction

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Bacterial strains and plasmids used are listed in Supplementary Data 1. E. coli strains were grown at 37 °C in LB medium^{57 (link)}. When required, E. coli cells were grown in M63 minimal medium⁵⁸ using the necessary nutritional supplements and 30 mM glycerol (Sigma, St. Louis, MO) as a carbon source. Antibiotics were added at the following concentrations: 100 μg ml⁻¹ ampicillin/carbenicillin and 25 μg ml⁻¹ chloramphenicol (Sigma, St. Louis, MO). For protein expression from pCKTRBS/pCKTRBS-OD plasmids, cultures were induced with 0.5 μg ml⁻¹ anhydrotetracycline (aTc) (Clontech, Mountain View, CA). When experimental conditions required, 1 mM IPTG (Sigma, St. Louis, MO), 1 mM sodium benzoate (Sigma, St. Louis, MO) and 0.4% glucose (Teknova, Hollister, CA) were supplemented.

Juárez J.F., Lecube-Azpeitia B., Brown S.L., Johnston C.D, & Church G.M. (2018). Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators. Nature Communications, 9, 3101.

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Induction Optimization for Bacterial Proteins

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All reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise noted. Antibiotics and IPTG were obtained from Gold Biotechnology (St. Louis, MO, USA). Anhydrotetracycline (aTC) was obtained from Clontech (Mountain View, CA, USA). Polymerase chain reaction (PCR) mix was purchased from Kapa Biosystems (Wilmington, MA, USA). Erythromycin and aTC were dissolved in ethanol while naringenin was dissolved in dimethyl sulfoxide. All other inducers were dissolved in deionized water.

Rogers J.K., Guzman C.D., Taylor N.D., Raman S., Anderson K, & Church G.M. (2015). Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Research, 43(15), 7648-7660.

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Bacterial Genetic Circuit Selection

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Ampicillin (100 μg/ml), kanamycin (25 μg/ml), chloramphenicol (12.5 μg/ml), and erythromycin (2 μg/ml) were used to select E. coli with genetic circuits in the pSB1A2, pSB3K3 and pSB4C5, and engineered B. subtilis, respectively. The Vibrio fischerii autoinducer N-3-oxohexanoyl-l-homoserine lactone (VAI, #K3007, Sigma Aldrich), Pseudomonas aeruginosa autoinducer (PAI, #O9139, Sigma Aldrich), Isopropyl-β-d-1-thiogalactopyranoside (IPTG, #I1284, Sigma Aldrich), anhydrotetracycline (aTc, #631310, Clontech), and tetracycline (Tc) were used as biosensor inputs. Sodium alginate (#W201502, Sigma Aldrich) and gelatin (#G9391, Sigma Aldrich) were used as hydrogel components. Catechol (#C9510, Sigma Aldrich) was used at 10 mM for XylE yellow staining.

Usai F., Loi G., Scocozza F., Bellato M., Castagliuolo I., Conti M, & Pasotti L. (2022). Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities. Materials Today Bio, 18, 100526.

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Growth Conditions for Bacterial Strains

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All strains were grown with aeration in Luria-Bertani (LB-Miller, BD-Difco) broth at 37°C except for the E. coli lambda cI857 lysogen which was grown at 30°C. Stra ins used in this study are listed in Table S1. Unless otherwise noted, antibiotics and inducers were used at: 100 μg mL⁻¹ ampicillin (Amp, Sigma), 100 μg mL⁻¹ kanamycin (Kan, GoldBio), 5 μg mL⁻¹ chloramphenicol (Cm, Sigma), 100 ng mL⁻¹ anhydrotetracycline (aTc, Clontech), and 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG, Fisher). For microscopy, HALO-TMR (Promega) and SNAP-JF₅₀₃ (Lavis Lab) were used at concentrations of 1 and 2 μM, respectively. AB solid agar consisted of 1.5% agar, 0.3 M NaCl, 50 mM MgSO₄, 0.2% casamino acids, 1 mM arginine, 1% glycerol, and 10 mM potassium phosphate at pH 7.5.

Silpe J.E., Bridges A.A., Huang X., Coronado D.R., Duddy O.P, & Bassler B.L. (2020). Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip. Cell host & microbe, 27(4), 629-641.e4.

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Mycobacterium tuberculosis Growth Conditions

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M. tuberculosis H37Rv and mihF-cKD strains were grown at 37 °C either in Middlebrook 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase, 0.2% glycerol and 0.05% Tween 80 or in Sauton’s liquid medium supplemented with 0.005% Tween 80. Cultures were plated on Middlebrook 7H10 (Difco) agar supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.2% glycerol. Hygromycin (50 μg ml⁻¹), kanamycin (25 µg ml⁻¹), streptomycin (25 μg ml⁻¹), 2.5% sucrose or Anhydrotetracycline (ATc, Clontech, 600 ng ml⁻¹) were added when needed. For cloning procedures, One shot® TOP10 chemically competent Escherichia coli (Invitrogen) were grown in Luria–Bertani (LB) broth or on LB agar with Hygromycin (200 μg ml⁻¹), kanamycin (50 μg ml⁻¹) or spectinomycin (25 μg ml⁻¹). All chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Experiments involving M. tuberculosis have been carried out in a Biosafety Level 3 (BSL3) laboratory, according to the national and international guidelines (Authorization number A070027/3).

Odermatt N.T., Sala C., Benjak A, & Cole S.T. (2018). Essential Nucleoid Associated Protein mIHF (Rv1388) Controls Virulence and Housekeeping Genes in Mycobacterium tuberculosis. Scientific Reports, 8, 14214.

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Bacterial Growth and Induction Conditions

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E. coli strains were grown with aeration in Luria–Bertani (LB-Miller, BD-Difco) broth. Aeromonas sp. ARM81 and V. fischeri strains were grown in LB with 3% NaCl. All strains were grown at 30 °C. Strains used in the study are listed in SI Appendix, Table S1. Unless otherwise noted, the following antibiotics and concentrations were used: 100 μg mL⁻¹ ampicillin (Amp, Sigma), 50 μg mL⁻¹ kanamycin (Kan, GoldBio), and 5 μg mL⁻¹ chloramphenicol (Cm, Sigma). Inducers were used as follows: E. coli: 200 μM isopropyl beta-D-1-thiogalactopyranoside (IPTG, GoldBio), 0.1% L-arabinose (Sigma), and 50 ng mL⁻¹ or 25 ng mL⁻¹ anhydrotetracycline (aTc, Clontech) and Aeromonas sp. ARM81: 0.1 ng mL⁻¹ aTc. HSL AIs were supplied at a final concentration of 20 μM, unless otherwise indicated.

Silpe J.E., Duddy O.P, & Bassler B.L. (2022). Natural and synthetic inhibitors of a phage-encoded quorum-sensing receptor affect phage–host dynamics in mixed bacterial communities. Proceedings of the National Academy of Sciences of the United States of America, 119(49), e2217813119.

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Genetic Modification and Virulence Testing of T. gondii

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The RH Δhxgprt (RH), TATi [66 (link)], DiCre-T2A [47 (link)] and RH Δku80 strains were used to construct genetically modified strains. All parasite strains were maintained in human foreskin fibroblast (HFF) cells (purchased from ATCC, USA), which were cultured in DMEM medium (Life Technologies, USA) containing 2% fetal bovine serum and 1% penicillin−streptomycin (Life Technologies, USA). Anhydrotetracycline (ATc) (Takara Bio USA, Inc., USA) at final concentration of 1 μg/mL was used to suppress the expression of pS1O7 regulated genes. Final concentration of 50 nM rapamycin (Aladdin, China) was used to induce excision of target genes engineered in the DiCre-T2A system. 10 mM mevalonate was used to induce IPP/DMAPP synthesis via the MVA pathway in the iTPI2compMVA strain. Seven-week-old female ICR mice were used for virulence tests of T. gondii strains. Nine-week-old Kunming mice were used to produce polyclonal antibodies against PGK2.

Niu Z., Ye S., Liu J., Lyu M., Xue L., Li M., Lyu C., Zhao J, & Shen B. (2022). Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth. PLOS Pathogens, 18(11), e1011009.

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Microbial Strain Characterization and Growth Conditions

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V. harveyi strains were derived from V. harveyi BB120 (BAA-1116) [62 (link)]. A. fischeri strains were derivatives of A. fischeri ES114 [63 (link)]. V. cholerae strains were derived from V. cholerae C6706str2 [64 (link)], and V. parahaemolyticus strains were derived from V. parahaemolyticus BB22OP (LM5312) [65 (link)]. E. coli BW25113 was used for heterologous gene expression and E. coli S17–1 λpir was used for cloning. All strains are listed in S1 Table. Vibrio and Aliivibrio strains were grown at 30°C shaking in either Luria Marine (LM) medium or minimal Autoinducer Bioassay (AB) medium, the latter supplemented with 0.4% vitamin-free casamino acids (Difco) [4 (link), 66 ]. E. coli strains were grown shaking at 37°C or at 30°C in LB medium. Antibiotics were added as follows (μg mL^-1): ampicillin, 100; chloramphenicol, 10; kanamycin, 100; polymyxin B, 50; and tetracycline, 10. Induction of genes on plasmids was accomplished by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Thermo Fisher), 0.2% arabinose (Sigma), or 100 ng mL^-1 anhydrotetracycline (aTc) (Takara), as necessary.

Eickhoff M.J., Fei C., Huang X, & Bassler B.L. (2021). LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA. PLoS Genetics, 17(4), e1009336.

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Propagation of Transgenic Parasite Strains

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The RH-Δhxgprt (RH) and TATi strains were propagated in human foreskin fibroblast (HFF) cells (purchased from the ATCC, Manassas, VA, USA) as described before (40 (link)). All other transgenic lines were constructed from these two strains and are described in more detail below. Anhydrotetracycline (ATc) (TaKaRa Bio USA, Inc., Mountain View, CA, USA) at a final concentration of 0.5 μg/ml was used to deplete PYK1 expression in all iPYK1-based strains.

Xia N., Ye S., Liang X., Chen P., Zhou Y., Fang R., Zhao J., Gupta N., Yang S., Yuan J, & Shen B. (2019). Pyruvate Homeostasis as a Determinant of Parasite Growth and Metabolic Plasticity in Toxoplasma gondii. mBio, 10(3), e00898-19.

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