CLONING OF BACILLUS THURINGIENSIS CRY3 FRAGMENT IN ESCHERICHIA COLI

(Published in Journal of Institute of Science and Technology, Vol. 15, 2007-08, pp.64-70)

Cloning of Bacillus thuringiensis cry3 fragment in Escherichia coli

Shyam K. Shah1, Kiran Babu Tiwari1,2,3 Upendra Thapa Shrestha2, Subarna Pokhrel4 and Vishwanath P. Agrawal1,2*
1Department of Biochemistry, Universal Science College, Pokhara University, Kathmandu, Nepal;
2Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal;
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal;

4School of Chemical and Biological Engineering, Seoul National University, South Korea
*Correspondence Address: : vpa@wlink.com.np.

Abstract

Bacillus thuringiensis was isolated and purified from the soil sample collected in Khumbu region of Mount Everest base camp. Total DNA was extracted and PCR was done using nine universal primers (Un1 to Un9) for cry1 to cry9 genes. A specific band of about 300 base pairs was amplified with universal primer Un3. DNA library was prepared into Escherichia coli HB101 using pUC18 vctor and HindIII restriction site. Upon PCR screening of 1000 clones using Un3 primer, three clones possessed cry3 gene. The cry3 specific fragment was cloned, extracted and purified. As the bacterium was isolated from high altitude, the gene may have novel biological function.