Cloning and expression of the gene encoding putative DEAD box helicase from Streptomyces coelicolor in homologous and heterologous host

Kovačević, Lucija (2013) Cloning and expression of the gene encoding putative DEAD box helicase from Streptomyces coelicolor in homologous and heterologous host. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

Helicases are essential enzymes that participate in nucleic acid metabolism. In this thesis we explored nucleotide sequence encoding a putative RNA (DEAD box) helicase (GI in NCBI: 6689196; gene annotation in StrepDB: SCO2863) from Streptomyces coelicolor A3(2). In the genome of this bacterium five potential genes encoding for RNA helicases are found, but none of these had been studied. In separate PCRs two fragments of the selected gene were cloned following the ligation of fragments into complete nucleotide sequence which was verified by sequencing. A construct containing the complete nucleotide sequence was cloned into expression vectors for helicase biosynthesis in homologous (S. coelicolor) and heterologous (Escherichia coli) hosts. The level of gene expression was studied by SDS–PAGE electrophoresis. Analysis revealed protein presence in the insoluble fraction of three different E. coli strains. Special genetic construct was used to transform protoplasts of S. coelicolor and DEAD box helicase was confirmed in the cell lysate by Western blotting method.

Item Type: Thesis (Diploma thesis)
Keywords: Streptomyces, bacteria E. coli, RNA helicase, gene cloning, protein biosynthesis
Supervisor: Vujaklija, Dušica
Date: 2013
Number of Pages: 48
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 19 Feb 2014 13:19
Last Modified: 22 Sep 2014 11:44
URI: http://digre.pmf.unizg.hr/id/eprint/441

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