The PURExpress Δ Ribosome kit was purchased from New England Biolabs (Cat no. E3313S). The components used to prepare Lysogeny Broth (LB Medium) for ribosome isolation were obtained from BD Biosciences and all other chemicals used were sourced from Merck Sigma Aldrich. (35S) Methionine was purchased from Perkin Elmer. Bis-Tris gels and plasmid isolation kits were obtained from Thermo Scientific.
Purexpress δ ribosome kit
Manufactured by New England Biolabs
The PURExpress® Δ Ribosome Kit is a cell-free protein synthesis system that allows for the production of proteins without the need for a cellular extract. The kit provides the necessary components for in vitro protein synthesis, including tRNAs, amino acids, and energy sources, but excludes ribosomes, which are the cellular structures responsible for protein synthesis. This allows for the study of protein synthesis in a simplified and controlled environment.
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Lab products found in correlation
Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Plasmid isolation kit, by Thermo Fisher Scientific (1 mentions)
Lysogeny broth medium, by BD (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nano glo luciferase assay kit, by Promega (1 mentions)
Rnasin, by Promega (1 mentions)
Whatman, by Cytiva (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Nupage 10 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Rnasecure, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by New England Biolabs (1 mentions)
Puromycin, by Bio-Rad (1 mentions)
Precision plus protein prestained standards, by Bio-Rad (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Tran35s label, by MP Biomedicals (1 mentions)
Amicon ultra 0.5 ml centrifugal filter, by Merck Group (1 mentions)
Purexpress in vitro protein synthesis system, by New England Biolabs (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
E coli ribosome, by New England Biolabs (1 mentions)
15 protocols using purexpress δ ribosome kit
1
Ribosome Isolation and Protein Labeling
2
In vitro Translation of NanoLuc Luciferase
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In vitro translation of NanoLuc was performed using a PURExpress ΔRibosome kit (NEB) as previously described (11 (link)). Briefly, 2.0 μl of solution A, 0.6 μl of factor mix, and 18 ng of DNA template (containing the T7 promoter, a 5′ untranscribed region [UTR] with a Shine-Dalgarno sequence, and the NanoLuc coding sequence) were mixed with 12.5 nM purified 70S ribosomes in a total volume of 5 μl. After 1 h of incubation at 37°C, relative luminescence units (RLU) produced from the translated luciferase was measured with a Nano-Glo luciferase assay kit (Promega Inc.) according to the manufacturer’s instructions. The inhibition of translation activity of 70S ribosomes purified from designated strains was assayed by adding the designated antibiotic at specified concentrations in the reaction mixture prior to ribosome addition and measuring luciferase activity after 1 h of incubation at 37°C. A reaction mixture without the antibiotic was used as a reference to determine percent inhibition. The IC50 value from a dose-response curve was obtained from nonlinear regression of log10 inhibitor versus activity plot using the variable slope model of GraphPad Prism software. The 95% confidence interval for each best-fit curve, also computed by Graph-Prism software from the standard errors, represents 95% probability that the computed IC50 value is in the given range.
3
In Vitro Synthesis of ApdA and ApdP
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To generate the ApdA-SRC, the ApdA mRNA template (500 ng μL−1) was translated by incubation in a B. subtilis in vitro translation system. Briefly, a total reaction volume of 450 μL was prepared by mixing 5.85 μL reconstitution buffer, 7.65 μL of methionine, 90 μL amino acid mix, 76.5 μL reaction mix (from RTS 100 HY Kit from Biotechrabbit GmbH) with 90 μL B. subtilis S12 translation extract, 135 μL ApdA mRNA, 45 μL 100 mM magnesium acetate, and then incubated for 40 min at 30 °C shaking in a thermomixer (500 rpm).
To generate the ApdP-SRC, the ApdP mRNA template (250 ng μL−1) was translated by incubation in a fully reconstituted E. coli in vitro PURExpress (NEB) translation system. Briefly, a total reaction volume of 100 μL was prepared by mixing 18 μL water, 40 μL solution A, 12 μL factor mix, 15 μL E. coli ribosome (PURExpress® Δ Ribosome Kit, NEB) with 15 μL ApdP mRNA, and then incubated for 40 min at 30 °C shaking in a thermomixer (500 rpm).
To generate the ApdP-SRC, the ApdP mRNA template (250 ng μL−1) was translated by incubation in a fully reconstituted E. coli in vitro PURExpress (NEB) translation system. Briefly, a total reaction volume of 100 μL was prepared by mixing 18 μL water, 40 μL solution A, 12 μL factor mix, 15 μL E. coli ribosome (PURExpress® Δ Ribosome Kit, NEB) with 15 μL ApdP mRNA, and then incubated for 40 min at 30 °C shaking in a thermomixer (500 rpm).
4
Cell-free Protein Synthesis Assay
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The PURExpress ΔRibosome kit (E3313S, New England Biolabs, Inc.) provided the basic materials for cell-free protein synthesis. In all translation experiments, the kit components (excluding ribosomes) were assembled according to the manufacturer's recommended concentrations. Purified SNAPf enzyme was purchased as SNAP-tag purified protein (P9312S, New England Biolabs, Inc.), and all fluorogenic SNAP substrates were provided by New England Biolabs as lyophilized powders, which were reconstituted to 1 mM in DMSO. The CBG-549-QSY7 substrate consists of guanine conjugated to fluorophore DY549P1 at the C6 position and to quencher QSY7 at C8, while CBG-488-TQ2 contains fluorophore ATTO488 and quencher TQ2.
5
Luminescence-based translation inhibition assay
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A luminescence based translation inhibition assay was performed using an in-vitro translation PURExpress® Δ Ribosome Kit from (NEB) with the pMSR plasmid, having the nLuc gene. The constituents of the kit were incubated with 50 ng/µl pMSR DNA template, 1U µl−1 murine ribonuclease inhibitor (thermo scientific), 2.4 µM crude ribosomes, with 1X and 2X molar higher concentration of RafH (wild type) or its mutants, W96A or W111A, and 5X Spectinomycin (at 5X it shows a similar inhibition as RafH protein), at 37 °C for 3 h. A total of eight reactions in triplicates with 10 µl reaction volume were incubated, then the reaction was quenched by keeping the reaction mixture on ice for 10 min. The luminescence, relative luminescence unit (RLU), was measured immediately after adding 30 µl NanoGlo substrate by using a GLOMAX luminometer from Agilent Technology (Promega). The data was plotted using GraphPad prism 8.0.1. (Fig.1e ).
6
Translation Assay of hlgCB and hlgB
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The translatability of hlgCB and hlgB mRNAs was assessed using the PURExpress Δ ribosome kit (NEB) supplemented with purified S. aureus 70S ribosomes (59 (link)). The ribosomes were first reactivated by incubation at 37°C for 10 min. The reactions were performed for 4 h at 37°C with various plasmids (200 ng of pUT7::hlgCB for the USA300, ST80, and PEN strains and pUT7::hlgB for the ST80 strain) and S. aureus 70S (30 pmol) in a solution containing 5 μL of buffer A, 1.5 μL of factors, 1 μL of RNasin (Promega), and 1 μL of [35S]methionine. Laemmli buffer was added to the samples, which were loaded onto an SDS–15% PAGE gel, with the Page Ruler Plus protein product (Thermo Fisher) run in parallel. After migration, the gel was heat-vacuum transferred onto Whatman paper and exposed to autoradiography film.
7
Fluorescence-based Protein Synthesis Assay
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PrimeSTAR HS DNA Polymerase was obtained from Takara Bio, Inc. The PURExpress ΔRibosome Kit, nuclease-free water and HaloTag TMR Ligand were purchased from New England Biolabs, QIAGEN and Promega, respectively. SUPERase-In RNase Inhibitor, NuPAGE 10% Bis-Tris Gel, NuPAGE MOPS SDS Running Buffer and RNAsecure were purchased from Life Technologies. Molecular weight markers (Precision Plus Protein Prestained Standards) and puromycin were purchased from Bio-Rad and Sigma-Aldrich, respectively. Anti-mouse IgG conjugated with HiLyte Fluor 555 [Anti-IgG (H + L), Mouse, Rabbit-Poly, HiLyte Fluor 555] was obtained from AnaSpec, Inc. Other reagents were purchased from Wako Pure Chemicals Industries, Ltd.
8
Recombinant Protein Purification and RNC Preparation
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Proteins (EF-G, G–II–III, and III–IV–V) were recombinantly expressed in E. coli BL21 cells and purified by affinity chromatography, followed by size exclusion chromatography. RNCs (531RNC) were prepared using the PURExpress Δribosome kit (NEB) supplemented with in-house purified ribosomes and programmed with mRNAs generated by in vitro transcription. Experimental procedures for the preparation of proteins and RNCs are described in detail in SI Appendix .
9
In Vitro Synthesis of mEGFP
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IC50 values of group A streptogramin analogs were determined using the PURExpress® Δ Ribosome Kit (E3313, NEB) for in vitro protein synthesis, 70S E. coli ribosomes (P0763S, NEB), murine RNAse inhibitor (M0314, NEB), and 6.66 ng/μl of template DNA encoding the fluorescent protein mEGFP (gifted by the Cate lab). This kit was specifically used to achieve a final ribosome concentration of 24 nM. The volume of the reaction mixture was scaled down 5-fold from the NEB protocol for a final reaction volume of 5 μL. Analogs were tested in a range from 0 to 36 μM in 10% DMSO (final concentration). Translation reactions were carried out in triplicate in a 37°C water bath for 4 hours, then transferred to a 0°C metal block. To assist in the transfer of reactions to 96-well half-area NBS microplates (Corning 3993) for final measurements, the reaction volume was increased to 50 μL by adding buffer (20 mM Tris-HCl pH 7.5, 60 mM NH4Cl, 6 mM MgCl2, 0.5 mM EDTA). Using a Cytation 5 plate reader (BioTek), translated mEGFP was excited at 485 nm; its emission was recorded at 535 nm. Raw data were processed and visualized using Python 2.7 and Matplotlib 2.0.2; the script is available on github. The IC50 was interpreted by fitting the dose response curve to the following equation, where Top and Bottom are the values of the plateaus: Y = Bottom + (Top - Bottom) / (1 + (X / IC50)).
10
In vitro Translation of GFP
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In vitro translation was performed using PURExpress® ΔRibosome Kit (New England Biolabs). Wild-type ribosomes (controls) or eluted ribosomes carrying E. coli or virus-encoded tagged bS21 were tested (final concentration: 10 pmol). A green fluorescent protein was translated by using a PCR product containing T7 promoter to rapidly evaluate the translation rates.
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