purexpress in vitro protein synthesis kit  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation in E. coli

doi: 10.1093/nar/gkaf1262

Figure Lengend Snippet: The change in mRNA stability is governed by the ribosome. (A) The in vivo fluorescence increase correlated with the S 0 or F 0 populations (normalized on the yfp mRNA signal at 10 min after induction). (B) Correlation of the in vivo or in vitro fluorescence increase with the yfp mRNA signal at 10 min after induction (normalized on the yfp mRNA signal). (C) Standard model of mRNA decay and our proposed model including the various variables used to construct the model. (D) Correlation of the logarithm of the in vivo fluorescence increase with the energy of folding (∆G) of the first 21 nucleotides calculated using the BindOligoNet G 5 model . In panels (A), (B), and (D) the red line is the linear regression, the r 2 of the goodness of the fit is reported in the right left corner of each graph. In panel (D) the black lines show the 95% confidence interval.

Article Snippet: To test the binding affinity of the TBT and TBT G mRNA to the 30S ribosomal subunit we used PURExpress ∆Ribosome Kit (NEB) supplemented with 5 μM of 30S ribosomal subunit and 10 μM tRNA fMet in the presence of 1.4 μM of radioactively labeled mRNA (prepared as described above by in vitro translation followed by [ 32 P] labeling as described for the northern blot probe labeling).

Techniques: In Vivo, Fluorescence, In Vitro, Construct

Journal: Nucleic Acids Research

Article Title: Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation in E. coli

doi: 10.1093/nar/gkaf1262

Figure Lengend Snippet: Change in A content for the TBT construct affects the formation of the 70S IC ribosome but not its affinity for the 30S subunit. (A) Filter binding assay of the P 32 -labeled mRNA (TBT or TBT G ), quantified after different incubation time with the 30S ribosomal subunit. Image filter after scanning are presented in the circles. (B) Polysomes separation and fractionation of IVTA programed with the TBT or TBT G mRNAs after 1 h of incubation (the IVTA is presented in the insert). (C) EtBr-stained gel or northern blot (N.B.) of the total RNA purified from each fraction of the polysome profiles. (D) Normalized graph of the quantification of the northern blot signal for the TBT or TBT G mRNAs. (E) Normalized graph of the quantification of the EtBr-stained gel signal for the 16S rRNA.

Article Snippet: To test the binding affinity of the TBT and TBT G mRNA to the 30S ribosomal subunit we used PURExpress ∆Ribosome Kit (NEB) supplemented with 5 μM of 30S ribosomal subunit and 10 μM tRNA fMet in the presence of 1.4 μM of radioactively labeled mRNA (prepared as described above by in vitro translation followed by [ 32 P] labeling as described for the northern blot probe labeling).

Techniques: Construct, Filter-binding Assay, Labeling, Incubation, Fractionation, Staining, Northern Blot, Purification

Journal: Nucleic Acids Research

Article Title: Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation in E. coli

doi: 10.1093/nar/gkaf1262

Figure Lengend Snippet: The change in mRNA stability is governed by the ribosome. (A) The in vivo fluorescence increase correlated with the S 0 or F 0 populations (normalized on the yfp mRNA signal at 10 min after induction). (B) Correlation of the in vivo or in vitro fluorescence increase with the yfp mRNA signal at 10 min after induction (normalized on the yfp mRNA signal). (C) Standard model of mRNA decay and our proposed model including the various variables used to construct the model. (D) Correlation of the logarithm of the in vivo fluorescence increase with the energy of folding (∆G) of the first 21 nucleotides calculated using the BindOligoNet G 5 model . In panels (A), (B), and (D) the red line is the linear regression, the r 2 of the goodness of the fit is reported in the right left corner of each graph. In panel (D) the black lines show the 95% confidence interval.

Article Snippet: For the IVTA separated on sucrose gradient reactions were performed in a final volume of 100 μl using the PURExpress ∆Ribosome In vitro Protein Synthesis Kit (New England Biolabs) according to the manufacturer instructions and with ribosomes purified from E. coli strain MRE600 [ ] at a final concentration of 2 μM.

Techniques: In Vivo, Fluorescence, In Vitro, Construct

Journal: Nucleic Acids Research

Article Title: Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation in E. coli

doi: 10.1093/nar/gkaf1262

Figure Lengend Snippet: Change in A content for the TBT construct affects the formation of the 70S IC ribosome but not its affinity for the 30S subunit. (A) Filter binding assay of the P 32 -labeled mRNA (TBT or TBT G ), quantified after different incubation time with the 30S ribosomal subunit. Image filter after scanning are presented in the circles. (B) Polysomes separation and fractionation of IVTA programed with the TBT or TBT G mRNAs after 1 h of incubation (the IVTA is presented in the insert). (C) EtBr-stained gel or northern blot (N.B.) of the total RNA purified from each fraction of the polysome profiles. (D) Normalized graph of the quantification of the northern blot signal for the TBT or TBT G mRNAs. (E) Normalized graph of the quantification of the EtBr-stained gel signal for the 16S rRNA.

Article Snippet: For the IVTA separated on sucrose gradient reactions were performed in a final volume of 100 μl using the PURExpress ∆Ribosome In vitro Protein Synthesis Kit (New England Biolabs) according to the manufacturer instructions and with ribosomes purified from E. coli strain MRE600 [ ] at a final concentration of 2 μM.

Techniques: Construct, Filter-binding Assay, Labeling, Incubation, Fractionation, Staining, Northern Blot, Purification

Journal: Poultry Science

Article Title: Isolation and characterization of bacteriocin-producing E. coli isolates from a poultry slaughterhouse, and cell-free production and evaluation of native and engineered bacteriocins

doi: 10.1016/j.psj.2025.105986

Figure Lengend Snippet: Antimicrobial activity of selected native bacteriocins synthesized using an in vitro cell-free protein synthesis (IV-CFPS) protocol. Activity was evaluated by the spot-on-agar-test (SOAT) against selected Gram-negative indicator strains. Shown halos correspond to representative results; all assays were performed in triplicate, and quantitative inhibition data (mean ± standard deviation) are provided in . NA: No activity detected.

Article Snippet: Forward and reverse primers were designed in accordance with the specifications of the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs), incorporating the T7 promoter and transcription terminator sequences.

Techniques: Activity Assay, Synthesized, In Vitro, Inhibition, Standard Deviation