Autori: Corcionivoschi N, Telea. A., Pacala N, Alemka A

Editorial: Romanian Biotechnological Letters 14., 2009.

Rezumat:

Our protocol to generate a null mutant involved the replacement of the cytochrome P450
coding sequence with an antibiotic resistance gene (kanamycin) by homologous recombination. Two
fragments, one upstream (F1 – 447 bp) and one downstream (F2 – 589 bp) of the P450 coding
sequence were amplified separately by PCR and combined during the second PCR reaction giving a
SmaI site at the junction point of the two DNA fragments. All the PCR reactions were performed using
the proofreading polymerase for amplification (PFU). The final DNA fragment obtained (F3 – 1036 bp)
was cloned into the SmaI site of the pBluescript SK- vector. This paper describes a new efficient
strategy of gene knockout in Campylobacter jejuni via kanamycin gene replacement.

Cuvinte cheie: campylobacter, gene deletion