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    Molecular phylogeny based on its sequences of nrDNA of some species belonging to dodder (Cuscuta L.) genus from various ecological sites of Turkey
    (Academic Press, 2020) Demir, İbrahim; Kaya, İlhan; Usta, Mustafa; Sipahioǧlu, Hikmet Murat
    Nuclear ribosomal DNA (nrDNA) sequence data of the Cuscuta genus, which have been considered as one of the most popular sequences for phylogenetic inference in plants, have been studied from a phylogenetic perspective in agricultural and non-agricultural lands of Turkey. The samples of Cuscuta spp. were collected from different geographical regions of Turkey between the years of 2013–2015. Some other species, not available locally, were taken from the herbarium samples of some research units. In order to study the phylogenetic relations of collected species, DNA isolations were made from body tissue samples. Conserved regions on ribosomal DNA (rDNA) were amplified by universal primers via PCR method and cloned into a proper cloning vector. The cloned DNA fragments were sequenced and analysed by web-based and computer programs. DNA sequences of certain species were recorded to the National Center for Biotechnology Information (NCBI) database. Based on the morphological examination and molecular analyses of fresh and the herbarium specimen, 8 species were identified. The identified species were C. hyalina (Gene bank accession no. KY020420), C. monogyna (KY020421), C. europaea (KY020422), C. palaestina (KY020423), C. approximata (KY020424), C. kurdica (KY020427), C. kotschyana (KY020430) and C. babylonica (KY020431). The ITS (Internal Transcribed Spacer) region contains several indels in identified Cuscuta species with the length varying from 668 to 730 bp. Sequence divergence ranges from 1.00% to 8.00% within Cuscuta spp. Based on our findings, the ITS sequences provided phylogenetically informative results in combination with the secondary structures.
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    Simultaneous production of alpha and beta amylase enzymes using separate gene bearing recombinant vectors in the same Escherichia coli cells
    (TÜBİTAK, 2020) Özcan, Dilek; Sipahioǧlu, Hikmet Murat
    The present study describes the simultaneous expression of thermostable industrial alpha (alpha) and beta (beta) amylase enzymes that have been used widely in starch industry. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for alpha amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for beta amylase were used as gene sources. Both genes were ligated into pETDuet-1 expression vector separately and resulting recombinant vectors were transformed into Escherichia coli BL21 competent cells by electroporation. The cells were first transformed by pETDuet-1/ alpha Amy recombinant plasmid, then the competent cells carrying this plasmid were prepared for the transformation of pETDuet-1/beta Amy plasmid. Enzymatic activities of bacterial colonies were detected on LB agar staining with iodide. Both enzymes were more produced by IPTG induction in BL21 cells and were purified using Ni-NTA agarose column. SDS-PAGE and western blot analyses showed that the molecular weight of purified alpha and beta amylase to be approximately 60 kDa and 55kDa, respectively. The concentration of the purified alpha and beta amylase were calculated as 4.59 mu g/mL and 3.17 mu g/mL with IPTG as an inducer in LB medium. The present study proposes a novel and efficient method for the production of thermostable alpha and beta amylases at the same E coli cells containing separate engineered plasmid vectors.