Cloning, expression T4 DNA ligase gene (T4Dnl) in E.coli host and purification of T4 DNA Ligase Enzyme

Ghahari, S. (2014) Cloning, expression T4 DNA ligase gene (T4Dnl) in E.coli host and purification of T4 DNA Ligase Enzyme. Masters thesis, University of Zabol.

Cloning, expression T4 DNA ligase gene (T4Dnl) in E.coli.pdf

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During the last 50 years, major advances in molecular biology have been attributed to the discovery of enzymes that allow molecular cloning of important genes. One of these enzymes that has been widely noteworthy for its role in the development of biotechnology is T4 DNA ligase. This enzyme have applications such as, cloning fragments into either plasmid or phage vectors, Joining of double-stranded oligonucleotide linkers or adaptors to DNA, Site directed mutagenesis, Amplified fragment length polymorphism (AFLP), Nick repair in duplex DNA, RNA or DNA/RNA hybrids, Self-circularization of linear DNA, and Ligase-mediated RNA detection. In this study, expression vectors pTrcHis-T4 and pET28-T4 separately transferred in competent Escherichia coli bacteria strains DH5α, TOP10F' and BL21 (DE3). The six samples were able to grow on selective medium, and after the confirmation of transformation, colons containing T4 DNA ligase gene was used to express recombinant protein. Final concentration of 1 mM IPTG inducer was used in step of recombinant protein expression. Then, 5 h after induction, the cells deposited and the cells lysised in the step of purification. Recombinant proteins containing histidine sequences were purified by nickel-agarose column manufacture by Qiagene Company. Finally, the purity rate of recombinant proteins was evaluated using the SDS-PAGE gel. The results showed that the purity of recombinant protein is over 90% and the most expression T4 DNA ligase recombinant protein for vector pET28-T4 was related to the Escherichia coli strain BL21 (DE3) and for vector pHis-T4 was related to E. coli strain TOP10F'.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Cloning; Expression; Protein Purification
Subjects: Q Science > QH Natural history > QH301 Biology
Depositing User: admin admin1 admin2
Date Deposited: 24 Apr 2016 04:32
Last Modified: 24 Apr 2016 04:32

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