S studyPrimers rex-F-HindIII rex-R-Xbal cydA-F cydA-RcydB-F cydB-RCon-F Con-R 16S rRNA-F 16S
S studyPrimers rex-F-HindIII rex-R-Xbal cydA-F cydA-RcydB-F cydB-RCon-F Con-R 16S rRNA-F 16S rRNA-R rbL13-F rbL13-R Sequence 5′ 3′ CTAAGCTTTGTCCGCACTCGCCGAC CTTCTAGAATCCACATCGGATCGATCGG TATCGCACCGGCAAGCAG GAACTCCTGCACGATGCC GATCTGCCCACCTTCTGG CATGCCGACGCCGAAGTC CCGTGATTTTGTAGCCCTGG GGCCTACTTCACCTATCCTGC CCTACGAGCTCTTTACGCCC AGAAGCACCGGCTAACTACG GGCGTAGACCTTGAGCTTC GCTCGAAAAGGCGATCAAGSpinosad in fermentation broth was extracted and determined by HPLC as described [10]. Dry cell weight (DCW) was determined as described [29]. Glucose was measured by utilizing the dinitrosalicylic acid (DNS) strategy [30]. The experiments had been repeated three occasions.NADH and NAD+ extraction and determinationNADH and NAD+ had been extracted based on a earlier described system with some modifications [31]. 5 mL cell cultures had been collected, chilled on ice immediately, and centrifuged at 12000 g, four for 10 min. Then cell pellets have been immediately ground to powder inside a porcelain mortar, which was pre-cooled to -80 , under liquid nitrogen for 5 min. After that, NADH was extracted by the addition of 300 uL 0.2 mol/L NaOH. NAD+ was extracted by the addition of 300 uL 0.two mol/L HCl. Then the samples had been heated at 50 for 10 min and neutralized utilizing NaOH or HCl. Soon after neutralization, the samples had been centrifuged at 12000 g, four for 10 min. The supernatant was collected and stored at -80 till made use of. NADH and NAD+ inside the supernatant were determined utilizing NAD/NADH quantitation kit (Comin), in accordance with manufacturer’s guidelines. The kit is determined by an enzymatic cycling assay method.Enzyme activity assays20 mL cell cultures were collected, chilled on ice instantly, and centrifuged at 3000 g, four for 10 min. Cell pellets had been suspended in two mL Tris Cl buffer (100 mM, pH 7.2) and Coccidia Compound disrupted by sonication on ice for five min (pulse intensity 40 , pulse on for 10 s and off for 50s). Soon after centrifugation (12000 g, four for 30 min), the supernatant was utilised for enzyme assay. 6-phosphofructokinase (PFK) activity was determined as described [31]. Isocitrate dehydrogenase (ICDH) activity was determined by measuring the production of NADH [32]. Glucose6-phophate dehydrogenase (G6PDH) activity was carried out by measuring the ACAT2 review formation of NADPH as described previously [33].Zhang et al. Microbial Cell Factories 2014, 13:98 microbialcellfactories.com/content/13/1/Page ten ofRNA extraction, cDNA synthesis, and real-time qPCR analysisExcellent Talents in University (NCET-10-0616) and All-natural Science Foundation of Tianjin (No. 10JCYBJC10300). Author particulars 1 Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. 2Key Laboratory of method bioengineering (Tianjin University), Ministry of Education, Tianjin 300072, PR China. 3Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, PR China. Received: eight March 2014 Accepted: 26 JuneRNA extraction, cDNA synthesis, and real-time qPCR evaluation of S. spinosa have been performed as described previously [34]. 16S rRNA and rbL13 had been used to normalize the qPCR data. The primers used in qPCR are listed in Table three.Intracellular metabolites employing GC-MS4 mL cell cultures had been mixed with six mL cold methanol (-40 ) to arrest metabolism instantaneously. Then, samples had been centrifugated at 3000 g for three min. Cell pellets have been collected and immediately ground to powder inside a porcelain mortar, which was pre-cooled to -80 , beneath liquid nitrogen for 5 min. Then.