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


Kovačević, Lucija
Cloning and expression of the gene encoding putative DEAD box helicase from Streptomyces coelicolor in homologous and heterologous host 2013., diplomski rad, diplomski, Prirodoslovno-matematički fakultet, Zagreb


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

Autori
Kovačević, Lucija

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, diplomski rad, diplomski

Fakultet
Prirodoslovno-matematički fakultet

Mjesto
Zagreb

Datum
11.02

Godina
2013

Stranica
48

Mentor
Dušica Vujaklija

Neposredni voditelj
Paradžik, Tina

Ključne riječi
Streptomyces bacteria E. coli ; RNA helicase ; gene cloning ; protein biosynthesis

Sažetak
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 (E. 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.

Izvorni jezik
Engleski

Znanstvena područja
Biologija



POVEZANOST RADA


Projekt / tema
098-0982913-2877 - Temeljna molekularno-biološka istraživanja streptomiceta (Dušica Vujaklija, )

Ustanove
Institut "Ruđer Bošković", Zagreb