Trimethoprim tmp

Manufactured by Merck Group

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Trimethoprim (TMP) is a synthetic antimicrobial compound used in various laboratory and pharmaceutical applications. It functions as a dihydrofolate reductase inhibitor, which is a key enzyme involved in the biosynthesis of folate. Trimethoprim can be utilized in assays, research, and development processes where its specific mode of action is relevant.

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Trimethoprim (207 citations) Trimethoprim/sulfamethoxazole (TMP/SMX) (2 citations)

9 protocols using trimethoprim tmp

Interferon Signaling Pathway Modulation

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Rhesus IFNα2 was obtained from R&D systems. Human IFNα and universal type I IFN (uIFN) were obtained from PBL Assay Science. MG132 (Fisher Scientific) was dissolved in DMSO and used at the indicated concentrations for 16 hours. Control wells were treated with same concentration of DMSO without MG132. Trimethoprim (TMP; Sigma-Aldrich) was dissolved in DMSO and used 10 μM or less where indicated. Viral cultures were supplemented with fresh TMP every 24 hours. The following antibodies were used for detection of endogenous and viral proteins in western blot: anti-ISG15 F-9 (Santa Cruz), anti-ISG54/IFIT2 (Abcam), anti-Mx-1 (GeneTex), anti-STAT1 M22 (Santa Cruz), anti-phosphorylated STAT1 Tyr701 (Santa Cruz), anti-STAT2 C20 (Santa Cruz), anti-phosphorylated STAT2 Tyr690 (Cell Signaling Technology), anti-IRF9/ISGF3γ clone 6 (BD Biosciences), anti-GAPDH 6C5 (Santa Cruz), anti-p84 5E10 (GeneTex), anti-IRF1 H-205 (Santa Cruz), anti-IRF3 (Santa Cruz) and anti-FLAG M2 (Sigma-Aldrich). The monoclonal antibodies specific for SVV and VZV ORF63 (clone 63_6), ORF62 (clone 62_6) and ORF31 (clone 31C_8) have been previously described [35 (link)]. STAT2 C20 (Santa Cruz) was also used for immunofluorescence microscopy.

Verweij M.C., Wellish M., Whitmer T., Malouli D., Lapel M., Jonjić S., Haas J.G., DeFilippis V.R., Mahalingam R, & Früh K. (2015). Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms. PLoS Pathogens, 11(5), e1004901.

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Sparse Cre-Expressing Cortical Neurons

Cited 4 times

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This study was carried out in accordance with the recommendations of UK Animals (Scientific Procedures) Act 1986 under Home Office Project and Personal Licenses (project licenses 70/7818 and 70/9095). The protocol was approved by the UK Home Office. Transgenic mice were bred to express VSFP Butterfly 1.2 in cortical layer 2/3 pyramidal neurons under the intersectional control of TetO and Cre-recombinase [Figure 2B, “sparse PC line,” Rasgrf2-dCre; CaMK2A-tTA; Ai78 (Mayford et al., 1996 (link); Harris et al., 2014 (link); Madisen et al., 2015 (link))]. To induce sparse expression through stochastic re-stabilization of destabilized Cre, a titrated total dose of 2E-4 mg/kg Trimethoprim (TMP, Sigma) was given via multiple intra-peritoneal injections over 2 consecutive days as described in Song et al. (2017 (link)). For comparison, transgenic mice densely expressing chimeric VSFP-Butterfly in all pyramidal cells were used [“pan PC line,” CaMK2A-tTA; tetO-chiVSFP (Song et al., 2018 (link))].

Quicke P., Song C., McKimm E.J., Milosevic M.M., Howe C.L., Neil M., Schultz S.R., Antic S.D., Foust A.J, & Knöpfel T. (2019). Single-Neuron Level One-Photon Voltage Imaging With Sparsely Targeted Genetically Encoded Voltage Indicators. Frontiers in Cellular Neuroscience, 13, 39.

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Comprehensive Protocol for Cell Culture Media Preparation

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Roswell Park Memorial Institute (RPMI) 1640 medium, no glutamine (21870076), HEPES (15630), Gentamicin (15750), L-glutamine (25030-024), Albumax II (11021-029) and SYBR green I (S7567) were purchased from Gibco, Thermo Fisher Scientific. RPMI 1640 Medium, no isoleucine (R9014) was purchased from US Biologicals. Bovine Serum Albumin (BSA) Fraction V (MB046) was purchased from NZYTech. RPMI 1640 no methionine, no cysteine (R7513), Glucose (G6152), Saponin (47036), Spermine (S4264), Spermidine (S0266), Putrescine (D13208), L-Glutathione reduced (G4251), L-Homocysteine (69453) and Trimethoprim (TMP) were purchased from Sigma-Aldrich. S-Adenosylmethione-1,4-butanedisulfonate (NA58198) was purchased from Biosynth Carbosynth. Paraformaldehyde was purchased from Santa Cruz Biotechnology.

Marreiros I.M., Marques S., Parreira A., Mastrodomenico V., Mounce B.C., Harris C.T., Kafsack B.F., Billker O., Zuzarte-Luís V, & Mota M.M. (2023). A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency. Communications Biology, 6, 205.

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Genome Engineering with CRISPR-Cas9

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All restriction enzymes, Klenow polymerase and T4 ligase were acquired from Thermo Scientific, Q5 and Phusion polymerases T7 endonuclease I, and Gibson Assembly Master Mix were from New England Biolabs Inc. Oligonucleotides were purchased from Microsynth AG and Sigma-Aldrich Co. and their sequences are listed in the Supplementary Data. Dulbecco’s modified Eagle’s Medium, foetal bovine serum, Turbofect, Lipofectamine 2000, GlutaMAX, penicillin, streptomycin and puromycin were acquired from Thermo Fisher Scientific, and trimethoprim (TMP) from Sigma-Aldrich Co. (T7883).

Tálas A., Kulcsár P.I., Weinhardt N., Borsy A., Tóth E., Szebényi K., Krausz S.L., Huszár K., Vida I., Sturm Á., Gordos B., Hoffmann O.I., Bencsura P., Nyeste A., Ligeti Z., Fodor E, & Welker E. (2017). A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a ‘self-cleaving’ GFP-expression plasmid. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 24(6), 609-621.

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Transfecting HEK293 and U2OS Cells

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HEK293 (a kind gift from Tomi Mäkelä, University of Helsinki, Finland) and U2OS (ATCC, Manassas, VA, USA) cells were maintained in DMEM supplemented with 10% fetal calf serum, L-glutamine and Penicillin/Streptomycin. To transfect plasmid DNA into the cells, Fugene 6 HD (Promega, Madison, WI, USA) or JETOptimus (Polyplus, Illkirch, France) were used following instructions provided by the manufacturer. Where indicated, trimethoprim (TMP, Sigma, St. Louis, MO, USA) was added to stabilize DD-dCas9-VP192 protein.

Elbasani E., Falasco F., Gramolelli S., Nurminen V., Günther T., Weltner J., Balboa D., Grundhoff A., Otonkoski T, & Ojala P.M. (2020). Kaposi’s Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter. Viruses, 12(9), 952.

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Burkholderia thailandensis E264 Transposon Mutagenesis

Cited 1 time

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Burkholderia thailandensis E264 and transposon mutants disrupting the dbcA gene were acquired from the Manoil lab (University of Washington)¹ (Gallagher et al., 2013 (link)). E. coli cultures were grown in lysogeny broth (LB) medium (1% tryptone, 0.5% yeast extract and 1% NaCl). B. thailandensis was grown in LB or cation adjusted Mueller-Hinton broth 2 (MH2; typical final pH 7.3. Sigma-Aldrich). Antibiotics colistin, ampicillin (Amp) 100 μg/ml, kanamycin (Kan) 30 μg/ml (E. coli) or 100 μg/ml (B. thailandensis), tetracycline (Tet) 12.5 μg/ml, and trimethoprim (Tmp) 100 μg/ml were purchased from Sigma-Aldrich or VWR. Cultures were grown at 37°C unless otherwise indicated.

Panta P.R., Kumar S., Stafford C.F., Billiot C.E., Douglass M.V., Herrera C.M., Trent M.S, & Doerrler W.T. (2019). A DedA Family Membrane Protein Is Required for Burkholderia thailandensis Colistin Resistance. Frontiers in Microbiology, 10, 2532.

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Molecular Cloning and Transfection Protocol

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle Medium DMEM (Gibco), fetal bovine serum (Gibco), Turbofect, Qubit dsDNA HS Assay Kit, Taq DNA polymerase (recombinant), TranscriptAid T7 High Yield Transcription Kit, Platinum Taq DNA polymerase, 0.45 µm sterile filters and penicillin/streptomycin were purchased from Thermo Fischer Scientific. DNA oligonucleotides, trimethoprim (TMP) and GenElute HP Plasmid Miniprep kit were acquired from Sigma–Aldrich. ZymoPure Plasmid Midiprep, RNA Clean & Concentrator kit and Maxiprep kits were purchased from Zymo Research. NEBuilder HiFi DNA Assembly Master Mix and Q5 High-Fidelity DNA Polymerase were obtained from New England Biolabs Inc. NucleoSpin Gel and PCR Clean-up kit was purchased from Macherey-Nagel. SF Cell Line 4D-Nucleofector X Kit S were purchased from Lonza, Bioruptor 0.5 ml Microtubes for DNA Shearing from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA universal qPCR Master Mix was purchased from KAPA Biosystems.

Kulcsár P.I., Tálas A., Ligeti Z., Krausz S.L, & Welker E. (2022). SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e. Nature Communications, 13, 6858.

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Antibiotics Evaluation in E. coli

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Unless otherwise stated, E. coli strains used in this study were grown in lysogeny broth (LB; Difco) or on LB agar plates. Broth cultures were incubated at 37 °C under shaking at 250 r.p.m. for 16 h, and LB agar plates in a 37 °C incubator for 16 h. Ceftriaxone (CRO), tetracycline (TET) and trimethoprim (TMP) were purchased from Sigma Aldrich. Gentamicin (GEN) and levofloxacin (LEV) were purchased from Biobasic and Santa Cruz Biotechnology, respectively.

Gingras H., Dridi B., Leprohon P, & Ouellette M. (2018). Coupling next-generation sequencing to dominant positive screens for finding antibiotic cellular targets and resistance mechanisms in Escherichia coli. Microbial Genomics, 4(2), e000148.

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Antibiotic Susceptibility Testing of E. coli

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The British Society for Antimicrobial Chemotherapy (BSAC) method for antibiotic susceptibility testing was used to prepare standardised inoculum (Andrews 2001 (link), 2002 ) and for breakpoint values (i.e. antimicrobial threshold concentrations that define resistance/susceptibility). Antimicrobial susceptibility profiles of E. coli clinical isolates were obtained for amoxicillin (AMX) (Sigma), cefotaxime (CTX) (Sigma), chloramphenicol (CAP) (ThermoFisher), ciprofloxacin (CIP) (Fluka), meropenem (MEM) (Sigma), nitrofurantoin (NIT) (Sigma), and trimethoprim (TMP) (Sigma) using the disc diffusion method. Due to poor agar diffusion, the susceptibility of the clinical isolates to polymyxin E (PME) (Sigma) was determined using the minimum inhibitory concentration (MIC) method.

Ribeiro C.A., Rahman L.A., Holmes L.G., Woody A.M., Webster C.M., Monaghan T.I., Robinson G.K., Mühlschlegel F.A., Goodhead I.B, & Shepherd M. (2021). Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved. Archives of Microbiology, 203(5), 2541-2550.

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