Cloning in expression vector pET22b (+)

Purified plasmid containing cellulase gene and pET22b (+) were restricted with SacI and NdeI restriction enzymes. Restriction of the plasmid was checked with the help of 1% agarose gel electrophoresis and desired DNA fragments were purified using MONARCH DNA Gel Extraction Kit Protocol (NEB #T1020) using the manufacturer’s guide. Purified restricted cellulase was then ligated into linearized pET22b (+). The ligation mixture was incubated on ice for 1 h and then at 20 °C for overnight. Competent cells of E. coli BL21 (DE3) were then transformed with this ligation mixture containing desired heterologous plasmid by conventional heat shock method. For the screening of transformants, the transformed cells were spread over the LB-agar plates (1.5% agar containing 50 μg ml⁻¹ ampicillin) and was incubated at 37℃ overnight. A single colony was isolated and inoculated in 10 ml of LB medium and 20 ml modified M9NG media, both containing 100 µg ml⁻¹ ampicillin. Both the flasks were incubated at 37 °C, 200 rpm on shaking incubator to grow overnight.