Journal: Translation

Article Title: Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

doi: 10.1080/21690731.2017.1327006

Figure Lengend Snippet: Compare PURE system transcription and translation with RTS 100 E. coli HY S30 system. (A) RNA denaturing gel of PURE reaction transcribing TC-Fluc mRNA. PURE system with no plasmid template and plasmid encoding TC-Fluc were incubated at 37°C for 2 h. Total RNA were purified by phenol/chloroform extraction and isopropanol precipitation from equal volumes of the 2 reactions and then run on lane 2 and 3, respectively. (B) RNA denaturing gel of RTS 100 E. coli HY S30 system transcribing TC-Fluc mRNA. RTS reactions with no plasmid template and plasmid encoding TC-Fluc were incubated at 30°C for 6 h. Total RNAs were purified by phenol/chloroform extraction and isopropanol precipitation from equal volumes of the 2 reactions and then run on lane 2 and 3, respectively. (C) Assessment of functional TC-Fluc translated in PURE and RTS 100 E. coli HY S30 systems. Equal volume aliquots were taken for luciferase assay to measure the actual amount of functional TC-Fluc produced. Values represent averages and error bars are ± standard deviations, with n = 3. (D) Assessment of full-length TC-Fluc produced in PURE and RTS 100 E. coli HY S30 systems. TC-Fluc synthesized in (C)were incubated with FlAsH-EDT2 biarsenical labeling reagent and analyzed on a SDS-PAGE. The gel was scanned by a typhoon scanner with filter set (508nmEx/528nmEm). Asterisk * indicates full-length product.

Article Snippet: 27 The genes encoding YaeJ and ArfA were cloned from E. coli MG1655 genomic DNA (ATCC) with a C-terminal histag and inserted into pET-24b (Merck Millipore) via NdeI and XhoI.

Techniques: Plasmid Preparation, Incubation, Purification, Extraction, Functional Assay, Luciferase, Produced, Synthesized, Labeling, SDS Page

Journal: Translation

Article Title: Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

doi: 10.1080/21690731.2017.1327006

Figure Lengend Snippet: Assessment of PURE and RTS 100 E. coli HY S30 system translation by production of HaloTag fusion proteins using linear DNA templates. HT-Ch-TolA, HT-EntE-Ch-TolA and HT-EntF-Ch-TolA with and without stop codon (82 kD, 141 kD, 224 kD) were synthesized in the PURE system and S30 extract and then incubated with HaloTag TMR Ligand. Equal volume aliquots of samples from PURE system and S30 extract reactions were analyzed on a SDS-PAGE and then scanned by a typhoon scanner with filter set (555nmEx/580nmEm). Lane 1, 3, 5, 8, 10, 12 are HaloTag fusion proteins with stop codon. Lane 2, 4, 6, 9, 11, 13 are HaloTag fusion proteins without stop codon. Lane 7 and 14 are negative controls with no template. (HT: HaloTag, 34 kD; Ch: mCherry, 29 kD. TolA, 19 kD, is a C-terminal 171-amino-acid α-helical spacer excised from E. coli TolA domain II. EntE, 59 kD, and EntF, 142 kD, are multidomain enzymes from E. coli enterobactin biosynthetic pathway.)

Article Snippet: 27 The genes encoding YaeJ and ArfA were cloned from E. coli MG1655 genomic DNA (ATCC) with a C-terminal histag and inserted into pET-24b (Merck Millipore) via NdeI and XhoI.

Techniques: Synthesized, Incubation, SDS Page

Journal: Nucleic Acids Research

Article Title: Detecting a wide range of epitranscriptomic modifications using a nanopore-sequencing-based computational approach with 1D score-clustering

doi: 10.1093/nar/gkae1168

Figure Lengend Snippet: Average F1-scores of the five algorithms compared in this study on the E. coli and S. cerevisiae rRNA test dataset ( NC : Nanocompore; DRM : Drummer; E - DSE: Epinano Delta-Sum-Error; E - LR : Epinano Linear Regression). The E. coli and S. cerevisiae rRNA datasets comprise 10 independent samples. Each sample contains eight subsamples with coverage-depths ranging from 10 to 2000. Different coverage-depths were used since algorithm performance depends on the coverage-depth, as indicated by recent studies ( , ) and also confirmed by our results. Note that all positions are treated as either positive or negative since unsupervised algorithms, do not distinguish between different modification types. In line with this, we do not compute separate F1-scores for each modification type separately, but rather only one F1-score for the whole dataset (for the given coverage-depth). As shown, Modena outperformed other algorithms across all coverage-depths; in some cases by a large margin (e.g. at coverage-depths of 50, 75, 100 and 200). The performance of all algorithms was very stable across the 10 independent samples . Thus, although the figure above shows average F1-scores, the results are highly consistent across all Samples 1–10.

Article Snippet: The E. coli K12 MG1655 strain was grown in 1× LB Broth Miller (1st Base, Singapore) without antibiotics at 37°C at 160 rpm shaking.

Techniques: Modification

Journal: Nucleic Acids Research

Article Title: Detecting a wide range of epitranscriptomic modifications using a nanopore-sequencing-based computational approach with 1D score-clustering

doi: 10.1093/nar/gkae1168

Figure Lengend Snippet: Precision–Recall curves (PR curves) for Sample 1 ( E. coli and S. cerevisiae rRNA dataset) for different coverage-depths. As shown, resampling increases the area under the PR curves (i.e. AUPRC scores) across all coverage-depths. Kuiper test further improves AUPRC scores across all coverage-depths, although to a lesser extent.

Article Snippet: The E. coli K12 MG1655 strain was grown in 1× LB Broth Miller (1st Base, Singapore) without antibiotics at 37°C at 160 rpm shaking.

Techniques:

Journal: Nucleic Acids Research

Article Title: Detecting a wide range of epitranscriptomic modifications using a nanopore-sequencing-based computational approach with 1D score-clustering

doi: 10.1093/nar/gkae1168

Figure Lengend Snippet: Violin plots of Modena score distributions for positive and negative test cases across different coverage-depths for Sample 1 of the E. coli / S. cerevisiae benchmark dataset are shown. Two well-separated clusters can be seen for all coverage-depths. The final Step 5 of our algorithm (1D score-clustering) leverages this separation to determine the classification threshold. Note that this represents a different paradigm from the standardly used P -value based thresholds. As shown in our study, this approach is not limited to Modena and can, in principle, be applied to any threshold-based unsupervised algorithm.

Article Snippet: The E. coli K12 MG1655 strain was grown in 1× LB Broth Miller (1st Base, Singapore) without antibiotics at 37°C at 160 rpm shaking.

Techniques:

Journal: Nucleic Acids Research

Article Title: Detecting a wide range of epitranscriptomic modifications using a nanopore-sequencing-based computational approach with 1D score-clustering

doi: 10.1093/nar/gkae1168

Figure Lengend Snippet: Average F1-scores (for Samples 1 through 10, E.coli / S. cerevisiae dataset) with coverage-depths ranging from 10 to 2000 are shown. Drummer : original Drummer algorithm with P -value and odds ratio-based threshold; Drummer + 1D clustering : Drummer algorithm (i.e. G-test statistic) with 1D score-clustering step (see Figure ). For detailed results across all samples, see and .

Article Snippet: The E. coli K12 MG1655 strain was grown in 1× LB Broth Miller (1st Base, Singapore) without antibiotics at 37°C at 160 rpm shaking.

Techniques:

Journal: Nucleic Acids Research

Article Title: Detecting a wide range of epitranscriptomic modifications using a nanopore-sequencing-based computational approach with 1D score-clustering

doi: 10.1093/nar/gkae1168

Figure Lengend Snippet: Average F1-scores (for Samples 1 through 10, E.coli / S. cerevisiae dataset) with coverage-depths ranging from 10 to 2000 are depicted. Epinano: Epinano-DSE algorithm with z-score based threshold; Epinano + 1D clustering : Epinano-DSE algorithm with 1D score-clustering step (see Figure ). For detailed results across all samples, see and .

Article Snippet: The E. coli K12 MG1655 strain was grown in 1× LB Broth Miller (1st Base, Singapore) without antibiotics at 37°C at 160 rpm shaking.

Techniques:

Journal: bioRxiv

Article Title: A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor

doi: 10.1101/2020.02.26.963322

Figure Lengend Snippet: a , The ter locus is organized in two operons, a putative biosynthetic cluster and a tellurium resistance cluster. These sections are found on opposite DNA strands and are encoded in bidirectionally. The representative ter locus from the hvKp strain NTUH-K2044 is shown. b, c , NTUH-K2044 containing an empty vector, the isogenic Δ terC mutant containing an empty vector, the pTerC, or the pTerZ-F plasmid ( b ), and the E. coli K12 strain MG1655 with or without the pTerZ-F plasmid ( c ) were grown on LB or LB containing 10 or 100 μM potassium tellurite to visualize inhibition of growth. Two representative clones of NTUH-K2044Δ terC containing the pTerC or the pTerZ-F plasmid and MG1655 containing the pTerZ-F plasmid are shown.

Article Snippet: E. coli K12 strain MG1655, Kp strain NTUH-K2044 , and isogenic mutants were cultured in Luria-Bertani (LB, Becton, Dickinson and Company, Franklin Lakes, NJ) broth at 37°C with shaking, or on LB agar at 27°C (Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Mutagenesis, Inhibition, Clone Assay