Cloning of ycjU from E. coli K-12 MG1655

The gene ycjU (gi|16129278; UniProt {"type":"entrez-protein","attrs":{"text":"P77366","term_id":"3024403"}}P77366) was amplified from the genomic DNA of E. coli K-12 MG1655 using the following set of primers:

5′-AATGCGGATCCATGAAACTGCAAGGGGTAATTTTCGATCTGG-3′

5′-ATAGTCTCGAGCTATACGTTTTGCCAGAAGGCCGATAACC-3′

The amplified gene was purified using the PCR cleanup kit and digested with the restriction enzymes BamHI and XhoI. The digested gene was ligated into a pET30a(+) vector, which was previously digested with the same two restriction enzymes and purified using the gel extraction kit. After verification of the gene sequence, E. coli BL-21 (DE3) cells were transformed with the plasmid and plated on LB agar.