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Saccharomyces cerevisiae
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The promoter fragment P8 in pGSR2518-4 was subcloned to identify the functional region of P8 (see Fig. 11). At first, to re-form the BamHl site at the 5' end of P8 in pGSR2518-4, the Sma l-Sac I fragment of pGSR2518-4 was exchanged with the Sma l-Sac I fragment of pGSR1518 containing the BamHl site to give the plasmid pGSR1518-4, which was subcloned with the P8 fragment. After each region between the BamHl site and the other unique restriction site (Nru I, EcoRV or Kpn I) in the P8 fragment was discarded from pGSR1518-4, each plasmid was used to transform S. cerevisiae YNN27. Consequently, only the transformant containing the plasmid in which the BamHl-Nru I region was discarded showed GSH A* activity. This plasmid and the P8 fragment without the BamHl-Nru I region were then designated pGSR1518-4d and AP8, respectively (see Fig. 11). The AP8 fragment and the 5'-flanking region of gsh A* in pGSR1518-4d were sequenced (Fig. 12). The AP8 fragment consisted of 1858 bp with 8 bp of the Xho I synthetic linker at the second nucleic acid of the translational initiation codon ATG, 391-bp downstream from the BamHl site of the AP8 fragment. The open-reading frame starting at this initiation codon was 3021 bp in length, corresponding to 1007 amino acids of 112,668 Da. As the transcriptional signals, two TATA boxes and two CAAT boxes were observed at positions -25 and -165 bp, and -248 and -278 bp, respectively, from the putative initiation codon ATG.
Synergistic polymorphic interactions of phase II metabolizing genes and their association toward lung cancer susceptibility in North Indians
Published in International Journal of Environmental Health Research, 2022
Harleen Kaur Walia, Parul Sharma, Navneet Singh, Siddharth Sharma
The PCR mixture of 25 µl was used to amplify the described fragment, which consists of 1X PCR buffer, 1.5 mM MgCl2, 0.5 µM of both forward and reverse primer, 200 µM dNTPs, 100 µg/ml bovine serum albumin (BSA) and 1 U Taq polymerase (DNAzyme, Thermo Scientific) and 200 ng DNA. After amplification, the PCR product of 162 bp for exon 3 variant and 357 bp for exon 4 variant was checked on 2.0% agarose gel and then digested with 5 U of EcoRV RsaI restriction enzyme (New England Biolabs, Ipswich MA), respectively at 37 °C. The digested samples were run on 8% Native-PAGE, stained in ethidium bromide, and visualized under a UV trans-illuminator. As described above, the amplified product was digested with their respective restriction enzymes. The digested products were resolved on either an agarose gel or a polyacrylamide gel to determine the restriction patterns. The genotypic status of the sample was determined by scoring the patterns. The genotyping of 20% of the samples was repeated twice to ensure that the results were reproducible, and it was found to be 100%.
A comparative study of the bispecific monoclonal antibody, blinatumomab expression in CHO cells and E. coli
Published in Preparative Biochemistry and Biotechnology, 2018
Fatemeh Naddafi, Farshad H. Shirazi, Yeganeh Talebkhan, Maryam Tabarzad, Farzaneh Barkhordari, Zahra Aliabadi Farahani, Elham Bayat, Reza Moazzami, Fereidoun Mahboudi, Fatemeh Davami
The expression cassette for blinatumomab was prepared in pcDNA3.1 (+) vector (Invitrogen). The sequence was sub-cloned from the pGH vector, into the pcDNA3.1 (+) vector by NheI/HindIII. Another expression cassette was FC550A-1, in which the sequence was cloned with an EcoRV restriction enzyme (Figure 1).