Rekombinant DNA teknolojisi ile Bacillus peptit feromonunun üretimi ve transformasyon etkinliğinin optimizasyonu
Production of Bacillus peptide pheromone with recombinant DNA technology and optimisation of transformation efficiency
- Tez No: 158251
- Danışmanlar: PROF. DR. FİLİZ ÖNER
- Tez Türü: Doktora
- Konular: Eczacılık ve Farmakoloji, Pharmacy and Pharmacology
- Anahtar Kelimeler: Rekombinant DNA teknolojisi, Escherichia coli, Bacillus Com X feromonu, klonlama, transformasyon, Recombinant DNA technology, Escherichia coli, Bacillus Com X pheromone, cloning, transformation
- Yıl: 2004
- Dil: Türkçe
- Üniversite: Hacettepe Üniversitesi
- Enstitü: Sağlık Bilimleri Enstitüsü
- Ana Bilim Dalı: Farmasötik Biyoteknoloji Ana Bilim Dalı
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 261
Özet
vıı ABSTRACT Demir D., Production of Bacillus peptide pheromone with recombinant DNA technology and optimisation of transformation efficiency, Hacettepe University, Health Sciences Institute PhD Thesis in Pharmaceutical Biotechnology, Ankara, 2004. With the advent of recombinant DNA technology bacteria are being genetically manipulated to produce products. By this way it is possible to modify organisms either by introducing foreign genes or by altering existing ones. The main strategy for introducing specific genes into host organisms, is transformation with a cloned vector-gene construct. In certain growth media Bacillus species express a set of proteins called, pheromones, involved in the transformation of foreign DNA. Genome of Bacillus species is well explained, Bacillus expression systems are non pathogenic, free of endotoxin, have high capacity for protein production but they are not used in the pharmaceutical applications extensively. In this theses study, Bacillus Com X pheromone genes were cloned into a plasmid vector so as to be expressed as a fusion protein. Prior to cloning studies desired PCR products for a 1 1 aa peptide of Bacillus mojavensis RO-B-2 strain ComX pheromone (GLQIYTNWVPS) were designed and amplified by using two complementary primers without any template DNA for the formation of primer- dimers. Taq polymerase amplified ComX PCR products were ligated directly without any modification by T4 DNA ligase into pGEM®-T Easy vector, which has a single overhanging T residue at the 3' end. Ligation mixture were transformed into Escherichia coli JM 109 and XL-1 Blue competent cells and clones were determined by ampicillin selection. Clones which have inserts are inframe with Lac Z promoter site to express fusion protein. Clones having inserts in the right or wrong position were determined by using PCR method, restriction endonucleases and DNA sequencing. Polyclonal antisera were raised against synthetic ComX pheromone peptide in the rabbits in order to be used in Western Blot analysis. Antibody titers were measured by using ELISA technique. Escherichia coli JM 109 and XL-1 Blue strains having ComX - pGEM®-T Easy vector construction were grown in liquid LB medium. Supernatants and inclusion bodies were isolated from growth medium andvi ayırt edildikten sonra, biyotinlenmiş füzyon protein içerikleri yönünden SDS-PAGE, Dot Blot ve Western Blot analizleri ile araştırılmışlardır. Jellerde ve nitroselüloz membranda beklenen yerde (6.5kDa) protein bantları gözlenmiştir. Bacillus mojavensis'in transformasyon etkinliği standart yöntem, sentetik peptit feromon ve füzyon protein ortamı karşılaştırılarak araştırılmış, sentetik peptit feromonun etki etmediği, füzyon protein ortamı ile ise transformasyonun arttığı görülmüştür. ı
Özet (Çeviri)
vıı ABSTRACT Demir D., Production of Bacillus peptide pheromone with recombinant DNA technology and optimisation of transformation efficiency, Hacettepe University, Health Sciences Institute PhD Thesis in Pharmaceutical Biotechnology, Ankara, 2004. With the advent of recombinant DNA technology bacteria are being genetically manipulated to produce products. By this way it is possible to modify organisms either by introducing foreign genes or by altering existing ones. The main strategy for introducing specific genes into host organisms, is transformation with a cloned vector-gene construct. In certain growth media Bacillus species express a set of proteins called, pheromones, involved in the transformation of foreign DNA. Genome of Bacillus species is well explained, Bacillus expression systems are non pathogenic, free of endotoxin, have high capacity for protein production but they are not used in the pharmaceutical applications extensively. In this theses study, Bacillus Com X pheromone genes were cloned into a plasmid vector so as to be expressed as a fusion protein. Prior to cloning studies desired PCR products for a 1 1 aa peptide of Bacillus mojavensis RO-B-2 strain ComX pheromone (GLQIYTNWVPS) were designed and amplified by using two complementary primers without any template DNA for the formation of primer- dimers. Taq polymerase amplified ComX PCR products were ligated directly without any modification by T4 DNA ligase into pGEM®-T Easy vector, which has a single overhanging T residue at the 3' end. Ligation mixture were transformed into Escherichia coli JM 109 and XL-1 Blue competent cells and clones were determined by ampicillin selection. Clones which have inserts are inframe with Lac Z promoter site to express fusion protein. Clones having inserts in the right or wrong position were determined by using PCR method, restriction endonucleases and DNA sequencing. Polyclonal antisera were raised against synthetic ComX pheromone peptide in the rabbits in order to be used in Western Blot analysis. Antibody titers were measured by using ELISA technique. Escherichia coli JM 109 and XL-1 Blue strains having ComX - pGEM®-T Easy vector construction were grown in liquid LB medium. Supernatants and inclusion bodies were isolated from growth medium andvııı analysed for their biotinylated protein content by using SDS-PAGE, Dot Blot and ;drpteee-(6.5 kBa)- on the gel and nitrocellulose membrane. Transformation efficiency of Bacillus mojavensis was compared by using standart method, synthetic peptide pheromone and fusion protein medium. Synthetic peptide pheromone did not affect transformation but fusion protein medium increased transformation efficiency.
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