Construction of pET-22b/f18 Recombinant Plasmid

The f18 gene was obtained from the genome of F18⁺ETEC (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"GQ325633.1","term_id":"261887612","term_text":"GQ325633.1"}}GQ325633.1) (Barth et al. 2011) by polymerase chain reaction (PCR) with specific primers F18F and F18R (Table 1). The pET22b plasmid was digested with NdeI and XhoI (Thermo Scientific, USA) to create sticky ends, followed by a recombinase-free cloning (RFC) protocol (Vo-Nguyen et al. 2022). The resulting mixture was transformed into E. coli DH5α competent cells. The transformants were initially screened on an ampicillin containing LB agar plate (LBAmp), then screened again by colony PCR with primers F18F/F18R and T7pro/T7ter. Plasmids collected from positive colonies were checked by the structure using digestion reaction with NdeI and XhoI, before sending for sequencing.

Primers used in this study.

Note: bold and underlined letters indicate restriction enzyme.