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

Year 2020, , 172 - 178, 31.12.2020

Ali Uslu , Osman Erganiş

https://doi.org/10.47027/duvetfd.825717

Abstract

Brusellozis, ülkemiz ve dünya hayvancılığına zarar veren kronik bakteriyel bir enfeksiyondur. Brucella doğada plazmidi bulunmadığından plazmid kaynaklı mutasyonlara çok açık değildir. Uzun pasajlar sonrası bakteriyel adaptasyon ile elde edilmiş mutantları bulunmaktadır. Konjugasyon, lipofectamin ve elektroporasyon ile yapılmış gen aktarım çalışmaları mevcuttur. Bu çalışmada kullanılan mutant pJQ200KS isimli intihar plazmidinde 6,7 kb (Backbone 5,4kb + 1,25kb İnsert) gibi oldukça büyük bir genoma sahiptir. Genom büyüdükçe transformasyonun zorlaşmaktadır. Sferoplast yapı, gen aktarımını kolaylaştırmada maya, mantar ve bitki hücrelerinde başarı ile kullanılmaktadır. Çalışmada Brucella’nın kompetent hale getirilerek gen aktarımının elektroporasyon ile kolaylaşması için hücreler, çeşitli yöntemlerle ile sferoplast haline getirildi ve gen aktarımında ki etkisine bakıldı. Stok Brucella melitensis Rev.1 kanlı agara pasajlandı. Kültür, Brucella broth içerisinde 37 ºC’de 24 saat boyunca 160 rpm’da inkübe edildi. Santrifüj ile 8000xg 4 ºC’de 5 dakika pelet haline getirildi, PBS ile yıkanarak besi yerinden arındırıldı. Sferoplast uyarımı için 72 saat boyunca penisilin, ampisilin ve glisin ile indüklendi. Elektroporasyon ile gen aktarımda en etkin yöntemin glisin ile uyarım metodu olduğu belirlendi. Aktarımı zor olan ve 7 kb’dan daha büyük olan plazmidlerin aktarımı için kompetent hücrenin sferoplast yapıya dönüştürülmesi metodu gelecekte yapılacak çalışmalarda araştırmacılara avantaj sağlayacağı düşünülmektedir.

Keywords

Brucella, Elektroporasyon, İntihar Plazmid, Sferoplast

Supporting Institution

Selçuk Üniversitesi

Project Number

2015-OYP-116 numaralı proje

Thanks

Elektroporasyon cihazı temini için Konya Gıda Tarım Üniversitesi KitArgem Birimine ve İstatiksel analiz için Doç.Dr. M.Agah Tekindal'a teşekkürü borç bilirim.

Making Brucella melitensis Rev.1 open to gene transfer with the help of spheroplast 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 conjugation. 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 cleared 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 studies.

Keywords

Brucella, Electroporation, Suicide Plasmid, Spheroplast

Project Number

2015-OYP-116 numaralı proje

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