Anhydrotetracycline

Manufactured by Takara Bio

Anhydrotetracycline is a chemical compound used in various laboratory applications. It serves as a key component in specific experimental procedures, but a detailed description of its core function would require more information to maintain an unbiased and factual approach.

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Lab products found in correlation

Fluoroskan ascent fl luminometer, by Thermo Fisher Scientific (2 mentions) Dual luciferase assay kit, by Promega (2 mentions) Facsaria 3 cell sorter, by BD (1 mentions) Vectashield hardset with dapi, by Vector Laboratories (1 mentions)

Close spelling variants of this product

Anhydrotetracycline (aTc,

7 protocols using anhydrotetracycline

Heterologous Expression of Tagged Proteins

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For the expression of FLAG-tagged CpoS proteins, the tag was inserted between the KpnI and SalI sites of p2TK2-SW2 (59 (link)). DNA fragments coding for CpoS proteins and their promoters were amplified (Table S3) or obtained as synthetic gene blocks (IDT) and then inserted between the AgeI and KpnI sites of the vector. For the expression of MYC-tagged Incs, the Inc genes and their promoters were amplified (Table S3) and then inserted between the AgeI and NheI sites of vector p2TK2-SW2 (59 (link)). Plasmids enabling the expression of mCherry (59 (link)) or inducible expression of GFP11-tagged IncB (CT232) (58 (link)) were gifts from Isabelle Derré and Kevin Hybiske, respectively. Bacteria were transformed as described above. Expression of GFP11-tagged IncB was induced by the addition of anhydrotetracycline (Clontech).

Meier K., Jachmann L.H., Türköz G., Babu Sait M.R., Pérez L., Kepp O., Valdivia R.H., Kroemer G, & Sixt B.S. (2023). The Chlamydia effector CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35. mBio, 14(4), e03190-22.

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Inducible Bacterial Gene Expression

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Overnight cultures of E. coli reporter strains were diluted 1:1000 in triplicate into M9 medium (0.5% glucose). Upon dilution, 100 ng mL⁻¹ anhydrotetracycline (Clontech) was added to induce qrr4 expression. Target-GFP translational fusions were induced by 0.5 mM IPTG. GFP fluorescence was measured after 8~10 h of growth using FACS (BD Biosciences FACSAria III cell sorter). Statistical analyses were performed using the unpaired t test, with a two-tailed p value < 0.05 considered significant.

Shao Y, & Bassler B.L. (2014). Quorum regulatory small RNAs repress type VI secretion in Vibrio cholerae. Molecular microbiology, 92(5), 921-930.

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Inducible fluorescent protein expression in Mycobacterium

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An extrachromosomal E. coli-mycobacteria shuttle vector, pMCSU7, was developed for the inducible expression of fluorescently tagged protein using the S. coelicolor tetO promoter from tcp3 (Additional file 5). The MadR1 coding sequence was PCR amplified from H37Rv genomic DNA and combined into pMCSU7 using Gateway technology. Constructs were screened using DNA sequencing prior to transformation into electrocompetent M. smegmatis. Mid-log grown transformants were diluted to O.D._600nm 0.2 and expression was induced in the dark for 6 hours using 50 ng ml⁻¹ anhydrotetracycline (Clontech). Cells were stained with FM 4-64 Fx in HBSS (Invitrogen™), fixed in 4% paraformaldehyde, applied to glass slides, and coated with Vectashield Hard Set with DAPI (Vector Laboratories). Slides were stored at 4C for a maximum of 24 hours before imaging at 1000x magnification using an inverted, oil-immersion Olympus IX71 microscope with a Retiga 2000R camera (QImaging) and Slidebook software (Intelligent Imaging Innovations Inc.).

Crew R., Ramirez M.V., England K, & Slayden R.A. (2015). MadR1, a Mycobacterium tuberculosis cell cycle stress response protein that is a member of a widely conserved protein class of prokaryotic, eukaryotic and archaeal origin. Tuberculosis (Edinburgh, Scotland), 95(3), 251-258.

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Plasmid Interference Assay with M13 Spacer

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The NCAs containing the M13 spacer were synthesized as gBlocks (IDT) and cloned by Gibson assembly into the pJKR-H-tetR vector (replacing the GFP gene downstream of the pLtetO promoter). Sequence-verified plasmids were transformed into E. coli K12 BW40114. A plasmid containing the M13 spacer target site was constructed by cloning the 33-bp target sequence into the pFN19K plasmid via PCR. A plasmid interference assay has been previously described^{24 (link)}. Briefly, overnight, cultures of strains containing the NCA plasmids were started from plates. In the morning, cultures were diluted in fresh LB containing the inducers arabinose, IPTG and anhydrotetracycline (Clontech), and grown for an additional 2 h. Cells were then washed three times in cold water, transformed with 50 ng pFN19K + M13 target plasmid and allowed to recover for ~1 h in LB at 37 °C before plating on LB plus Kan plates (absolute efficiency) and LB plus Carb plates (to normalize efficiencies).

Nivala J., Shipman S.L, & Church G.M. (2018). Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration. Nature microbiology, 3(3), 310-318.

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SaeS Phosphorylation Assay Protocol

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S. aureus strains harboring pYJ-saeRS were grown to exponential growth phase at 37°C and the SaeS protein was induced by the addition of anhydrotetracycline (Clontech, 0.5 μg/ml) at 37°C for an additional 4 h. Cell membranes were prepared as described previously [49 (link)]. The SaeS phosphorylation assay was carried out as described above except that the purified cell membranes (25 μg) were used as a source of SaeS and the incubation time was 10 min.

Cho H., Jeong D.W., Liu Q., Yeo W.S., Vogl T., Skaar E.P., Chazin W.J, & Bae T. (2015). Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections. PLoS Pathogens, 11(7), e1005026.

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Measuring Mistranslation Rates in Mycobacteria

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Mistranslation rates were measured in strains with M. tuberculosisgatCA-WT, G444S, and K61N constructed on an isogenic M. smegmatisgatCA deletion background with Renilla/firefly dual-luciferase reporters (9 (link)). The construct containing D120N in the Renilla luciferase sequence was used to measure the asparagine-to-aspartate mistranslation rate, while the construct containing K529R in the firefly luciferase sequence was used to measure the near-synonymous arginine-to-lysine mistranslation rate. The assay was carried out as previously described (7 (link), 29 (link), 31 (link)). Briefly, strains containing mistranslation reporters were grown to stationary phase (OD₆₀₀ > 3) and diluted into fresh 7H9 medium supplemented with 0/0.5 mM trehalose to a final OD₆₀₀ of ∼0.2. Expression of the reporter was induced with 100 ng/ml anhydrotetracycline (Clontech; catalog no. 631310) for 6 to 8 h. Bacteria were pelleted and lysed with passive lysis buffer provided in the dual-luciferase assay kit (Promega; catalog no. E1960), and luminescence was measured by a Fluoroskan Ascent FL luminometer (Thermo Scientific) according to the manufacturer’s instructions, with 1,000 ms as the integration time.

Li Y.Y., Cai R.J., Yang J.Y., Hendrickson T.L., Xiang Y, & Javid B. (2021). Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity. mBio, 12(4), e01100-21.

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Measuring M. tuberculosis Mistranslation Rates

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Mistranslation rates were measured in strains with M. tuberculosis gatCA-WT, G444S and K61N, constructed on an isogenic M. smegmatis gatCA deletion background with Renilla-Firefly dualluciferase reporters (9) . The construct containing D120N in the Renilla luciferase sequence was used to measure the asparagine-to-aspartate mistranslation rate, while the construct containing K529R in the Firefly luciferase sequence was used to measure the near-synonymous arginine-to-lysine mistranslation rate. The assay was carried out as previously described (7, 25, 26) . Briefly, strains containing mistranslation reporters were grown to stationary phase (OD600nm > 3) and diluted into fresh 7H9 medium supplemented with 0 / 0.5 mM trehalose to a final OD600nm ~0.2. Expression of the reporter was induced with 100 ng/mL anhydrotetracycline (Clontech, #631310) for 6-8 hours. Bacteria were pelleted and lysed with passive lysis buffer provided in the dual-luciferase assay kit (Promega, #E1960), and luminescence was measured by a Fluoroskan Ascent FL luminometer (Thermo Scientific) according to the manufacturer's instructions, with 1,000 ms as the integration time.

Li Y., Cai R., Yang J., Hendrickson T.L., Xiang Y., & Javid B. (2021). Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity.

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