Brucella melitensis Rev.1’in Sferoplast Yapısı ile Gen Transferine Açık Hale Getirilmesi

Abstract

Structure Abstract Brucellosis is a chronic bacterial infection that harms livestock in our country and the world. Because of Brucella does not have a natural plasmid, it is not very open to plasmid-derived mutations. However, there are Brucella mutants obtained after long passages with bacterial adaptation. Gene transfer studies with conjugation, lipofectamine and electroporation are available. The suicide plasmid called pJQ200KS used in this study cannot be accomplished by conjugation since it does not have the λ pir gene region required for conjugatio n. It has been reported that as the size of the genome used for transfer increases, transformation becomes more difficult. Spheroplast structure has been successfully used in yeast, fungi and plant cells to facilitate gene transfer. There are two studies conducted for the transformation of Brucella into spheroplast structure. In studies to obtain spheroplasts from Brucella , after 24 hours of pre-culture, the spheroplast cells were exposed to the stimulation of penicillin, ampicillin and glycine for 48 hours, and penicillin and glycine were taken as the most effective stimulation method. Stock Brucella melitensis Rev.1 was passaged on blood agar. The culture was incubated in Brucella broth at 37 ° C for 24 hours at 160 rpm. 8000xg was pelleted for 5 minutes at 4 ° C by centrifugation, and the medium was cleare d by washing with PBS. It was induced with penicillin, ampicillin and glycine for 72 hours for spheroplast stimulation. The structure has been made suitable for gene transfer. Since penicillin completely removes the cell wall structure of Brucella , unlike spheroplast formation, that it harms bacteria. It was determined that the most effective method in transferring with electroporation was stimulation with glycine. The method of transforming the component cell into a spheroplast structure for the transfer of plasmids that are difficult to transfer and larger than 7 kb will provide an advantage to researchers in