Journal: Mlife

Article Title: Optimizing a high‐sensitivity NanoLuc‐based bioluminescence system for in vivo evaluation of antimicrobial treatment

doi: 10.1002/mlf2.12091

Figure Lengend Snippet: Construction of stable and functional Nluc‐based luciferase‐producing Staphylococcus aureus reporter strains. (A) Western blot analysis of the expression of PBP2a, Eno, CobB, and SarA in S. aureus USA300 over time. The protein gel served as a loading control (Lc), and the molecular weights of the protein markers (M) are indicated on the left. (B) Semi‐quantitative analysis of the gray values of the indicated bands in each lane of (A) using ImageJ software. Data are presented as mean ± SEM. Statistical significance was calculated by two‐way analysis of variance (ANOVA); ns indicates no significance, ** p < 0.01, and *** p < 0.001. (C) Western blot analysis of Eno fusion proteins in the total cell lysates using mouse anti‐Eno polyclonal antibodies. (D) Bioluminescence (BL) photographs of different S. aureus reporter strain and substrate combinations. (E) Representative BL images of 50 µl of S. aureus reporter strains (1 × 10 7 CFU/ml) mixed with 50 µl of different doses of HFZ, FUR, or DTZ (3.125–100 µM). (F) Quantification of total flux produced from S. aureus reporter strains with different concentrations of substrates. Data are presented as mean ± SEM. Statistical significance was analyzed by two‐way ANOVA; ns indicates no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001. (G) Images of BL signals from USA300/Eno‐Nluc, USA300/Eno‐Teluc, and USA300/Eno‐Antares2 over a range of bacterial loads with 50 µl of HFZ (100 µM) in the black 96‐well plates. (H) Good correlation between the BL intensity and bacterial number of S. aureus USA300/Eno‐Nluc, USA300/Eno‐Teluc, or USA300/Eno‐Antares2. The experiment was repeated at least three times in triplicate. Data are presented as mean ± SEM. x , bacterial number; y , BL intensity.

Article Snippet: The nucleotide sequences of nluc, teluc , and antares2 according to the codon usage bias of S. aureus USA300 were chemically synthesized by BGI‐Shenzhen (China) and cloned into the pUC18 plasmid to achieve pUC‐ nluc , pUC‐ teluc , and pUC‐ antares2 , respectively (Table ).

Techniques: Functional Assay, Luciferase, Western Blot, Expressing, Control, Software, Produced

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: PCR amplification of three cellulase genes. Lanes : 1, marker; 2, cel7482; 3, control (ddH 2 O as template); 4, marker; 5, cel3623; 6, control; 7, marker; 8, cel36; 9, control

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Amplification, Marker

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: SDS-PAGE analysis for the expression of the cellulase genes in E. coli BL21 (DE3). Lane 1 , total proteins from recombinant E. coli BL21 cells without IPTG induction; lane 2 , total proteins from recombinant E. coli BL21 cells induced by IPTG for 4 h; lane 3 , purified cellulase; M, protein molecular weight markers. a cel7482; b cel3623; c cel36

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: SDS Page, Expressing, Recombinant, Purification, Molecular Weight

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: Purification of the recombinant His 6 -tagged cel7482, cel3623 and cel36 from 500 ml of E . coli cultures using immobilized metal ion affinity chromatography

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Purification, Recombinant, Activity Assay

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: HPAEC analysis for final products of hydrolysis of CMC by the recombinant cel7482, cel3623 and cel36. HPAEC analysis was performed on a Dionex ICS5000 system equipped with a pulsed amperometric detector and a CarboPac PA200 column (Dionex). a cel7482; b cel3623; c cel36

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Recombinant

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: a The effect of pH on activity of the recombinant cel7482, cel3623 and cel36. b The effect of temperature on activity of the recombinant cel7482, cel3623 and cel36

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Activity Assay, Recombinant

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: Thermostability of the recombinant cel7482, cel3623 and cel36. Each purified enzyme was incubated at different temperatures for different periods of time before determining the residual activity at optimal temperature in 50 mM citrate–phosphate buffer (pH 5.5) with CMC as substrate. a cel7482; b cel3623; c cel36

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Recombinant, Purification, Incubation, Activity Assay

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: a The effect of NaCl on the activity of the recombinant cel7482, cel3623 and cel36. Each purified enzyme was incubated with CMC at optimal temperature for 30 min in 50 mM citrate–phosphate buffer (pH 5.5) in the presence of 0.5–5 M NaCl. b Halotolerance of the recombinant cel7482. After pre-incubation in 0.5, 2 and 5 M NaCl for different periods of time, the residual activity was determined at 70 °C in 50 mM citrate–phosphate buffer (pH 5.5) with CMC as substrate

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Activity Assay, Recombinant, Purification, Incubation

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: a The predicted 3D structure of cel7482 ( side view and top view ) using the crystal structure of a family 5 endoglucanase (PDB: 1ceo) as modeling template. The colored sticks represent the conserved residues in the active site. The orange sticks represent the different residues between cel7482 and cel3623. b The predicted 3D structure of cel36 ( side view and top view ) using the crystal structure of endo-1,4-β-glucanase from Bacillus subtilis 168 (PDB: 3pzt) as modeling template. The colored sticks represent the conserved residues in the active site

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques:

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: Cellulase activities of the culture supernatant and total cell lysate derived from B. subtilis 168 and its tat mutant strains. 168, B. subtilis 168; tatAyCy , tat mutant strain lacking functional TatAyCy translocase; tatAdCd , tat mutant strain lacking functional TatAdCd translocase; total- tat 2 , tat mutant strain lacking all Tat translocases. For expression of YwbN-cel7482 fusion protein, the culture of the recombinant B. subtilis was induction with 0.5 % xylose at 37 °C for 24 h

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Derivative Assay, Mutagenesis, Functional Assay, Expressing, Recombinant

Journal: Biotechnology for Biofuels

Article Title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy

doi: 10.1186/s13068-016-0557-3

Figure Lengend Snippet: Strains, plasmids and primers used in this study

Article Snippet: The nucleotide sequence encoding the twin-arginine signal peptide of B . subtilis YwbN [ ] and codon-optimized cel7482 was chemically synthesized by BGI Inc., Beijing, China.

Techniques: Plasmid Preparation, TA Cloning, Sequencing, Expressing, Recombinant