S30 premix without amino acids

Manufactured by Promega

The S30 premix without amino acids is a laboratory reagent designed for in vitro protein expression. It provides the necessary components for cell-free protein synthesis, excluding amino acids. The premix contains the essential buffers, salts, and cofactors required to support the translation of mRNA into proteins.

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Lab products found in correlation

Flx800, by Agilent Technologies (2 mentions) Luciferase assay reagent, by Promega (2 mentions) Ribolock, by Thermo Fisher Scientific (1 mentions) Flx800 luminometer, by Agilent Technologies (1 mentions) Ribolock rnase inhibitor, by Promega (1 mentions) Luciferase assay substrate, by Promega (1 mentions) D300e digital dispenser, by Tecan (1 mentions) T7 rna polymerase, by Thermo Fisher Scientific (1 mentions) Rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Protease inhibitor, by Roche (1 mentions) Microfluidizer processor, by Microfluidics (1 mentions) Luciferase reporter assay system, by Promega (1 mentions)

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Amino acids (6 citations)

5 protocols using s30 premix without amino acids

Measuring Ribosomal Mistranslation In Vitro

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Purified bacterial 70S ribosomes were used in dual luciferase translation assays as previously described 48, 49. A typical translation reaction (30 μl) contained 0.25 μM 70S ribosomes, 4 μg firefly (F‐luc) and 0.4 μg Renilla (R‐luc) mRNA, 40% (vol/vol) of M. smegmatis S100 extract, 200 μM amino acid mixture, 24 units of RiboLock (Thermo Fisher), and 0.4 mg/ml of tRNAs—energy was supplied by the addition of 12 μl of commercial S30 Premix without amino acids (Promega). The reaction mixture was incubated at 37°C for 35 min, stopped on ice, and assayed for F‐luc and R‐luc luciferase activities according to supplier's protocol (Promega). Luminescence was measured using the FLx800 luminometer (BioTek Instruments).
Mistranslation was determined as described previously 50, 51. In brief, to assess misreading His245 (CAC codon) of the F‐luc gene was replaced by the near‐cognate codon CGC or the non‐cognate codon AGA, both encoding for Arg. To determine read‐through, the Asp357 (GAC codon) of the F‐luc gene was replaced by the nonsense TGA stop codon. Arg245F‐luc mRNA, X357TGA F‐luc mRNA, and WT F‐luc mRNA were used in in vitro translation reactions, and R‐luc mRNA was used as internal control. Mistranslation was calculated by the ratio of mutant firefly/Renilla luciferase activity to wild‐type firefly/Renilla luciferase activity.

Akbergenov R., Duscha S., Fritz A., Juskeviciene R., Oishi N., Schmitt K., Shcherbakov D., Teo Y., Boukari H., Freihofer P., Isnard‐Petit P., Oettinghaus B., Frank S., Thiam K., Rehrauer H., Westhof E., Schacht J., Eckert A., Wolfer D, & Böttger E.C. (2018). Mutant MRPS5 affects mitoribosomal accuracy and confers stress‐related behavioral alterations. EMBO Reports, 19(11), e46193.

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In Vitro Ribosomal Protein Synthesis Assay

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Purified 70S ribosomes and in vitro-transcribed firefly luciferase mRNA were used in translation reactions. Firefly luciferase mRNA was produced using T7 RNA polymerase in vitro. A typical translation reaction mixture contained 0.21 µM 70S ribosomes, 450 ng mRNA, 20% (v/v) M. smegmatis S100 extract, 200 µM amino acid mixture, 0.3 µL 50× protease inhibitor coctail (Roche) and Ribo Lock RNase inhibitor (12 units; Promega), 0.4 mg/ml tRNAs and 40% (v/v) commercial S30 premix without amino acids (Promega). 1.5 µL antibiotics (final reaction concentrations between 0.01 and 1000 µM) or water were added to the mixture. The total volume of the reaction mixture was 15 µL. The reaction mixture was incubated at 37°C for 30 min and stopped on ice. Finally, 75 µL luciferase assay substrate (Promega) was added and the bioluminescence measured in a luminometer (Flx800; Bio-Tek Instruments). The IC₅₀ is defined as drug concentration required to inhibit synthesis of active luciferase to 50%.

Freihofer P., Akbergenov R., Teo Y., Juskeviciene R., Andersson D.I, & Böttger E.C. (2016). Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase. RNA, 22(12), 1836-1843.

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Cell-free Ribosome Translation Inhibition Assay

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Cell-free in-vitro translation inhibition assays were performed using luciferase mRNA and bacterial S30 extracts containing either wild-type bacterial or human hybrid ribosomes.^{55 (link)} In brief, firefly luciferase mRNA was transcribed in vitro using T7 RNA polymerase (Thermo) using a plasmid as template in which the mammalian promoter in pGL4.14 (Promega) has been replaced by theT7 bacteriophage promoter. Test articles in aqueous solution containing 0.3% Tween20 were dispensed into white 96-well plates (Eppendorf) using the TECAN D300e digital dispenser. The test article dispension volume was balanced to a total of 1.5 μL by 0.3% Tween20 in water. The reaction volume was brought to 15 μL by addition of 13.5 μL Translation Master Mix comprised of bacterial S30 extract, 0.2 mM amino acid mix, 6 μg tRNA (Sigma), 0.4 μg hFluc mRNA, 0.3 μL protease inhibitor (cOmplete, EDTA-free, Roche), 12 U RNAse inhibitor (Ribolock, Thermo Scientific), and 6 μL S30 premix without amino acids (Promega). Dispension and mixing of reagents was performed on ice prior to incubating the sealed plates at 37 °C. After 30 minutes of incubation, the reaction was stopped on ice and 75 μL of luciferase assay reagent (Promega) was added to each well. Luminescence was recorded with a plate reader (BIO-TEK FLx800, Witec AG, Littau, Switzerland).

Lubriks D., Zogota R., Sarpe V.A., Matsushita T., Sati G.C., Haldimann K., Gysin M., Böttger E.C., Vasella A., Suna E., Hobbie S.N, & Crich D. (2021). Synthesis and Antibacterial Activity of Propylamycin Derivatives Functionalized at the 5”- and Other Positions with a View to Overcoming Resistance due to Aminoglycoside Modifying Enzymes.. ACS infectious diseases, 7(8), 2413-2424.

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Mycobacterial Cell-Free Protein Synthesis Assay

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Mycobacterial cell-free translation inhibition assays were performed as described previously (40 (link)). In brief, M. smegmatis strain SZ380 bacterial cells were disrupted and emulsified with a microfluidizer processor (Microfluidics, Westwood, MA, USA) at 25,000 lb/in². S30 extracts were prepared by addition of dithiothreitol (DTT) to 1 mM and centrifugation at 30,000 × g at 4 °C. Translation reaction mixtures containing either test article or dimethyl sulfoxide (DMSO) vehicle control and 4 µL of the S30 extract, 0.2 mM amino acid mix, 6 µg tRNA (Sigma), 0.4 µg hFluc mRNA, 0.3 µL protease inhibitor (complete, EDTA-free, Roche), 12 U RNAse inhibitor (Ribolock, Thermo Scientific), 6 µL S30 premix without amino acids (Promega), plus water to a final reaction volume of 15 µL were incubated for 1 h at 37 °C. The reaction was stopped on ice before adding 75 µL of luciferase assay reagent (Promega) and recording of luminescence. Regression analysis and IC₅₀ calculation were performed using GraphPad Prism version 9.3.1 by using the equation [log(inhibitor) vs. response–Variable slope (four parameters)] with the fitting method: least squares (ordinary) fit. Y = Bottom + (Top-Bottom)/(1+10^((X-LogIC50))).

Phelps G.A., Cheramie M.N., Fernando D.M., Selchow P., Meyer C.J., Waidyarachchi S.L., Dharuman S., Liu J., Meuli M., Molin M.D., Killam B.Y., Murphy P.A., Reeve S.M., Wilt L.A., Anderson S.M., Yang L., Lee R.B., Temrikar Z.H., Lukka P.B., Meibohm B., Polikanov Y.S., Hobbie S.N., Böttger E.C., Sander P, & Lee R.E. (2024). Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus. Proceedings of the National Academy of Sciences of the United States of America, 121(2), e2314101120.

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Inhibition of Cell-free Protein Synthesis by AGA

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Ribosomes and ribosome-free S100 cell extracts were purified from bacterial cell pellets as described previously using sucrose gradient (10%-40%, w/v) centrifugation. 17 Concentration of 70S ribosomes was determined by absorption measurements using a standard value of 23 pmol ribosomes per A260 unit. Firefly luciferase (F-luc) mRNA was produced in vitro using T7 RNA polymerase (Thermo Scientific Fermentas, Reinach, Switzerland) on templates of modified plasmid pGL4.14 (Promega, Mannheim, Germany). 17 A typical translation reaction with a total volume of 30 mL contained 0.25 mM 70S ribosomes, 4 mg of F-luc mRNA, 40% (v/v) of ribosome-free S100 extract, 200 mM amino acid mixture, 24 U of RNAse inhibitor (Ribo Lock, Thermo Scientific Fermentas), 0.4 g/L total E. coli tRNA and 12 mL of commercial S30 Premix without amino acids (Promega).
After addition of serially diluted AGA, the reaction mixture was incubated at 378C for 35 min and subsequently placed on ice. F-luc activity was quantified using the Luciferase Reporter Assay System (Promega). Luminescence was measured using a luminometer (FLx800; Bio-Tek Instruments, Luzern, Switzerland). IC 50 values represent the AGA concentrations that inhibit F-luc activity by 50%. Student's t-test was used where applicable. The level of significance was set at P values of ,0.05.

Maurer F.P., Bruderer V.L., Castelberg C., Ritter C., Scherbakov D., Bloemberg G.V, & Böttger E.C. (2015). Aminoglycoside-modifying enzymes determine the innate susceptibility to aminoglycoside antibiotics in rapidly growing mycobacteria. The Journal of antimicrobial chemotherapy, 70(5).

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