China
Africa
Explore chapters and articles related to this topic
Functional Metagenomics for Bioprospecting Novel Glycosyl Hydrolases in 2G Biofuel Production from Lignocellulosics
Published in Jitendra Kumar Saini, Surender Singh, Lata Nain, Sustainable Microbial Technologies for Valorization of Agro-Industrial Wastes, 2023
Sugitha Thankappan, Sajan Kurien, Surender Singh, Asish K. Binodh
The major limitation in the enrichment studies is the loss of relevance as the microbial populations subjected to enrichment undergo external modifications in the natural biomass ecosystem. The use of silica gel in metagenomic DNA extraction after cell lysis will reduce the shearing to some extent and enhance the mgDNA recovery as well as the quality. However, in the alternate strategy, the large particulate matter is separated by a gentle centrifugation technique (645 times gravity/3,000 rpm) before the mgDNA extraction. The separation step is followed by the addition of a lysis buffer containing CTAB, ethylene diamine, EDTA, NaCl, Tris-HCl, and proteinase K for microbial cell lysis. These strategies are generally employed for unveiling novel genes encoding hydrolytic enzymes, like cellulases, esterases, proteases, lipases, xylanases, etc.
1
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
Dan Shu, Yi Shu, Farzin Haque, Sherine Abdelmawla, Peixuan Guo
Primers for human GAPDH and Survivin are:GAPDH left: 5′-AGCCACATCGCTCAGACAC-3′;GAPDH right: 5′-GCCCAATACGACCAAATCC-3′;Survivin left: 5′-CACCGCATCTCTACATTCAAGA-3′;Survivin right: 5′-CAAGTCTGGCTCGTTCTCAGT-3′.PCR was performed on LightCycler® 480 for 45 cycles. The data were analyzed by the comparative CT Method (ΔΔCT Method). For Western Blot assays, cells were lysed by RIPA lysis buffer (Sigma) and the cell total protein was extracted for the assay. Equal amounts of proteins were then loaded onto 15% SDS-PAGE and electrophoretically transferred to Immun-Blot PVDF membranes (Bio-Rad). The membrane was probed with Survivin antibody (R&D) (1:4000 diluted) and β-actin antibody (Sigma) (1:5000 diluted) overnight, followed by 1:10000 anti-rabbit secondary antibody conjugated with HRP (Millipore) for 1 h. Membranes were blotted by ECL kits (Millipore) and exposed to film for autoradiography.
Growth of Mesenchymal Stem Cells on Surface-Treated 2d Poly(Glycerol-Sebacate) Bio-Elastomers of Varying Stiffness
Published in Jose James, Sabu Thomas, Nandakumar Kalarikkal, Yang Weimin, Kaushik Pal, Processing and Characterization of Multicomponent Polymer Systems, 2019
Raju B. Maliger, Peter J. Halley, Justin J. Cooper-White, Donna Dinnes
DNA assay: The DNA assay was performed in a 96-well black flat-bottomed plate (Perkin-Elmer). First, a DNA standard curve was prepared using calf thymus DNA (Sigma Aldrich, Sydney). 10 μL of lysis buffer was added to each well-containing DNA standard. This was done to compensate for the lysis buffer contained in the cell lysate samples. 10 μL of sample from each cell lysate was added to each sample well. Working DNA dye solution was prepared by mixing 1 mL 10 X TNE (100mM Tris; 2.0M NaCl; 10mM EDTA; pH 7.4), 9 mL ddH2O, and 10 μL Hoechst dye (1 mg/mL Hoechst stock). Then 100 μL of working DNA dye solution was added to each standard and sample well to be analyzed. The analysis was performed by using SpectraMax M5 (Molecular Devices) Platereader and setting UV excitation and emission wavelengths at 360 and 460 nm, respectively. The DNA assay was performed three times independently for every experimental sample.
Production of highly soluble native human paraoxonase 2 with potential anti-biofilm property
Published in Preparative Biochemistry & Biotechnology, 2023
Fauzia Parween, Priyamedha Yadav, Kalyani Singh, Rinkoo Devi Gupta
The primary culture was set from the transformed E. coli Rosetta (DE3) cells and grown overnight in the presence of kanamycin in LB broth for 14–16 hours at 37 °C. The primary culture was used to set up secondary culture and allowed to grow till O.D. 0.4–0.6 at 600 nm and 37 °C. Different concentrations of IPTG were used to induce the secondary culture and incubated at different temperatures (25 °C, 30 °C and 37 °C). 1 mM CaCl2 was also added to the secondary culture at the time of IPTG induction. The cells were harvested after 6 hours of induction by centrifugation at 4000 g and re-suspended in lysis buffer. The lysis buffer was made in 50 mM Tris–HCl pH 8.0, with 1.0 mg/ml lysozyme, Triton X-100 (0.5%), 1 mM CaCl2, and 1 mM PMSF. The cell lysate was sonicated at 30% amplitude, 5–6 times for 20 s each with intervals of 20 s. A clear solution was obtained. After centrifugation at 12,000 g, the pellet and supernatant were collected separately. An equal quantity of lysis buffer (without lysozyme) was used to re-suspend the pellet. Thereafter, the expression of HuPON2 was checked on SDS-PAGE in all the supernatants and pellets. Here, most of the protein was expressed in the pellet fraction (Figure S1B). To solubilize the protein, different buffers were used (Table 1). The expressed protein was resolved using 12% SDS-PAGE gel, and subsequently, the proteins were checked for its enzyme activity.
Construction of polysaccharide scaffold-based perfusion bioreactor supporting liver cell aggregates for drug screening
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Lei Cao, Huicun Zhao, Mengyuan Qian, Chuxiao Shao, Yan Zhang, Jun Yang
The cell viability of the aggregates in the bioreactor was assessed by fluorescein diacetate (FDA)-propidium iodide (PI) (St. Louis, MO, USA) staining. After the scaffolds were immersed in 2 mL 0.1 M sodium citrate (prepared with 0.9% NaCl) for 15 min, the aggregates were collected by centrifuged at 150 g for 5 min and washed 3 times with PBS. And then the aggregates were incubated in fresh WE containing 5 mg/mL FDA and 2 mg/mL PI at room temperature for 5 min. After being rinsed 3 times with PBS, the stained aggregates were observed immediately under a confocal scanning laser microscopy (Leica TCS SP8, Wetzlar, Germany) at 488 nm/561 nm. The number of cells was evaluated by the total protein collected from the scaffolds. To obtain the total protein of the cells, lysis buffer (10 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, and protease inhibitor cocktail, pH 7.4) was added to the scaffolds on ice. The lysates were centrifuged at 13 000 rpm for 10 min at 4 °C. The supernatants were collected, and the concentration of total protein was determined by a BCA assay kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. A standard curve was drawn according to the number of cells and total protein extracted.
Noncorrelative relation between in vitro and in vivo for plasmid DNA transfection by succinylated polyethylenimine muscular injection
Published in Journal of Biomaterials Science, Polymer Edition, 2021
Yuki Kobayashi, Kei Nirasawa, Yoichi Negishi, Shoichiro Asayama
In vivo transfection into the skeletal muscles of mice with bPEI-Et-COOH was performed using previously described methods [14–16]. Briefly, ICR mice (5 weeks old, male) were anesthetized with pentobarbital. The complexes between pDNA (5 µg) and bPEI-Et-COOH (5 µg, 10 µg or 25 µg) in 35 µL of PBS(-), which were incubated at 37 °C for 24 h, were injected into the tibialis muscles of the ICR mice. Seven days after the injection, the whole tibialis muscles were collected and homogenized. The tissue homogenates were prepared in lysis buffer (0.1 M Tris-HCl (pH 7.8), 0.1% Triton X-100, and 2 mM EDTA). Luciferase activity was measured using a luciferase assay system (Promega, Madison, WI) and a luminometer. The activity is indicated by relative light units (RLU) per mg of protein. The pcDNA3-Luc plasmid, derived from pGL3-basic (Promega, Madison, WI), was used as an expression vector encoding the firefly luciferase gene under the control of a cytomegalovirus promoter.