Escherichia coli top 10 chemically competent cells

A synthetic open reading frame for Bm86 antigen expressed in insect was assembled by GeneArt AG (Life Technologies) and cloned into pMK-RQ (kanR) using the cloning sites Sfil/Sfil. The purified plasmid DNA was transformed into Escherichia coli Top10 chemically competent cells (Invitrogen) to promote propagation and the transformed E. coli was cultivated overnight on LB agar plates containing 100 μg/mL ampicillin at 37°C. Single colonies conferring ampicillin resistance were selected and grown overnight in 10 mL of LB containing 100 μg/mL ampicillin with vigorous shaking at 37°C. Cells were harvested by centrifugation at 10,000 g, and pMK-RQBM86 vector was purified from the overnight culture using a Qiaprep spin miniprep kit (Qiagen). The 1890bp Bm86 fragment was released from the pMK-RQ vector by restriction endonuclease digestion, using BamHI and HindIII restriction enzymes (NEB) following manufacturer's instructions. The Bm86 gene fragment was successfully ligated into the pFASTBAC1 expression vector and transformed into top 10 chemically competent cells (Invitrogen), followed by overnight cultivation at 37°C on LB plates containing 100 μg/mL ampicillin. The final plasmid construct was verified by DNA sequencing to confirm the correct insertion of the Bm86 open reading frame.