“CLONING AND EXPRESSION OF GLUTATHIONE S-TRANSFERASE GENE IN

PROKARYOTIC SYSTEM”

Mamatha J1* ., Ishfaq Hassan., MirKhan .,Mohd Rijvan., Milton Mali.,Saikh Parvez &
Chetana S

*Email correspondence: mamathaj.raj@gmail.com

1* PG -Department of Biotechnology, The Oxford College of Science, (TOCS) Bangalore.

Glutathione S-Transferases (GST) represent a complex grouping of proteins widely

distributed in prokaryotes and are grouped into several classes. Bacterial GSTs are implicated
in a variety of distinct processes such as the biodegradation of xenobiotics, protection against
chemical and oxidative stresses and antimicrobial drug resistance and also involved in a
variety of distinct metabolic processes such as the biotransformation of dichloromethane, the
degradation of lignin and atrazine. In present study aimed at cloning and expression of GST
gene in E.coli K-12 substr. MG1655 . Genomic DNA from Escherichia coli K-12 substr.
MG1655 were isolated and purified using agarose gel electrophoresis. The concentration of
DNA was determined using a spectrophotometer at 260/280 nm. Primers for GST gene were
designed by Oligo Calc and NEB cutter software and its amplification by PCR techniques.
BamH1 and Xho1 restriction endonucleases were used to digest both pET-32b (+)
vector and GST gene . Ligation of GST B gene into pET-32b (+) vector were successful with
the help of T4 DNA Ligase followed by transformation of ligated GST B gene in to
Prokaryotic system. After transformation the plates were observed for analyzing
transformation efficiency. Two separate colonies were visualized on the agar plate. White
coloured colonies contained the gene of interest and thus were transformed, whereas the blue
colonies were deficit of the gene of interest and showed no transformation. Specific activity
for E.coli was found to be 31.25 µmole/ml/min.

Key words: Glutathione S-Transferases, E.coli, pET-32b(+) Vector, DNA Ligase.

Corresponding author address:

Mamatha J.

Associate Professor,

P.G. Department of Biotechnology,

The Oxford College of Science,

Bangalore- 560 102,

Karnataka, India Tel: +91 9738109917, Email: mamathaj.raj@gmail.com