Topraktan izole edilen Bacillus megaterium EBD 9-1 suşundan fitaz geninin E.coli'de klonlanma çalışmaları
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Date
2015-01-07
Authors
Zeren, Behice
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Publisher
Uludağ Üniversitesi
Abstract
Bu çalışmada Türkiye'nin 30 farklı ilinden temin edilen toprak örneklerinden 300 adet bakteri izole edilmiştir. Bakterilerin morfolojik ve fizyolojik özellikleri incelenmiş ve 236 bakteri Bacillus cinsi olarak tanımlanmıştır. Bütün suşların fitaz aktiviteleri fitaz tarama ortamı (PSM) kullanılarak tespit edilmiş ve açık zonların çapı mm olarak gösterilmiştir. Toplam 19 Bacillus sp. suşu, fıtat parçalayıcı bir enzim olarak bilinen ekstraselüler fitaz üreticisi olarak bulunmuştur. Bu suşlar arasında, en geniş zona (11 mm) sahip 1 adet Bacillus sp. suşu seçilmiş ve Bacillus sp. EBD 9-1 olarak adlandırılmıştır. 16S rRNA dizi analizi sonucunda Bacillus sp. EBD 9-1 suşunun Bacillus megaterium ile %100 benzerlik gösterdiği tespit edilmiş ve yeni izolat Bacillus megaterium EBD 9-1 olarak adlandırılmıştır. Bacillus megaterium EBD 9-1 suşunun fitaz enzim geni E.coli DH5α suşunda klonlanmaya çalışılmıştır. Bacillus megaterium DSM319, Bacillus megaterium QM B1551 ve Bacillus megaterium WSH-002 suşlarının bütün genom sekansı NCBI veri bankasında mevcuttur. Ancak, bu sekansların içinde fitaza ait bir sekans bulunamamıştır. Bu sebeple, farklı Bacillus türlerinin fitaz sekansları hizalanmış ve bir çifti dejenere olmak üzere 2 primer çifti hazırlanmıştır. PZR (Polimeraz Zincir Reaksiyonu) sonucunda, sadece dejenere primer çiftleri kullanılarak amplifikasyon gerçekleştirilmiş ve üç bant (~900 bp, ~500 bp) görülmüştür. Çoğaltılan bu DNA fragmanları pGEM®-T Easy vektörüne ligasyon ile aktarılmıştır. Rekombinant plazmitlerin E.coli DH5α suşuna transformasyonu sağlanmış ve rekombinant plazmidi içeren saf E.coli DH5α suşunun plazmidleri izole edilmiştir. Klonlanmanın kontrolü için, rekombinant plazmid EcoRI restriksiyon endonükleaz enzimi ile kesilmiş ve beklenen boydaki bantlar agaroz jel profilinde gözlenmiştir. İzole edilen plazmidler T7 primeri ve ABI PRISM-310 Genetic Analyzer cihazı kullanılarak sekanslamıştır. NCBI data bankası kullanılarak saptanan sekansın literatür analizi yapılmıştır. Elde edilen sonuçlara göre 3 DNA fragmanının nükleotit dizisinin fitaz enzimi sekansı ile benzerlik göstermediği belirlenmiştir. Dolayısıyla dejenere primerler ile amplifikasyon sonucunda fitaz geni çoğaltılamamıştır. Ancak, bu yeni izolat literatür için önemlidir. Yeni izole edilen bu Bacillus megaterium EBD 9-1 suşu endüstriyel ölçekte fitaz üretimi büyük bir potansiyele sahip olabilir.
In this study, 300 bacteria were isolated from soil samples that provided from 30 different cities of Turkey. Morphological and physiological features of bacteria were investigated, and 236 bacteria were defined as Bacillus genus. The phytase activities of all strains were assayed using phytase screening medium (PSM), and exhibited as diameter of clear zone in mm. Total 19 Bacillus sp. strains were found as extracellular phytase, known as phytate-degrading enzyme, producer. Among of these strains, one strain of Bacillus sp. that had the most largest zones (11 mm) was selected, and was named as Bacillus sp. EBD 9-1. 16S rRNA sequence analysis revealed that Bacillus sp. EBD 9-1 was found to be 100% similar with Bacillus megaterium, and new isolate was named as Bacillus megaterium EBD 9-1. Phytase gene of Bacillus megaterium EBD 9-1 strain was tried to be cloned into the E. coli DH5α strain. The whole genome sequence of Bacillus megaterium DSM319, Bacillus megaterium and Bacillus megaterium QM B1551 Bacillus megaterium WSH-002 strains are available at NCBI data bank. However, was not found any sequence of the phytase in these sequences. Therefore, phytase sequences of different Bacillus species were aligned, and two primer pair with one degenerate was prepared. In PCR (Polymerase Chain Reaction) result, it was realized amplification just using the degenerate primer pairs, and three bands (~900 bp, ~500 bp) were observed. The amplified DNA fragments were transferred into pGEM®-T Easy vector by ligation. Recombinant plasmids were transformed into E.coli DH5α strain, and plasmids of pure E. coli DH5α strains containing the recombinant plasmid was isolated. For control of cloning, the recombinant plasmid was cut with EcoRI restriction endonuclease enzyme, and bands of the expected size were observed in the agarose gel profile. The isolated plasmids were sequenced using T7 primer and the ABI PRISM-310 Genetic Analyzer apparatus. Literature analysis of the sequence determined using the NCBI data bank has been made. According to the results, the nucleotide sequence of all three DNA fragments was determined not show sequence similarity with phytase enzyme. So with, phytase gene as a result of amplification with degenerate primers was not amplified. However, these new isolate is important for literature. This newly isolated Bacillus megaterium sp. EBD 9-1 strain may be great potential for phytase production at industrial scale.
In this study, 300 bacteria were isolated from soil samples that provided from 30 different cities of Turkey. Morphological and physiological features of bacteria were investigated, and 236 bacteria were defined as Bacillus genus. The phytase activities of all strains were assayed using phytase screening medium (PSM), and exhibited as diameter of clear zone in mm. Total 19 Bacillus sp. strains were found as extracellular phytase, known as phytate-degrading enzyme, producer. Among of these strains, one strain of Bacillus sp. that had the most largest zones (11 mm) was selected, and was named as Bacillus sp. EBD 9-1. 16S rRNA sequence analysis revealed that Bacillus sp. EBD 9-1 was found to be 100% similar with Bacillus megaterium, and new isolate was named as Bacillus megaterium EBD 9-1. Phytase gene of Bacillus megaterium EBD 9-1 strain was tried to be cloned into the E. coli DH5α strain. The whole genome sequence of Bacillus megaterium DSM319, Bacillus megaterium and Bacillus megaterium QM B1551 Bacillus megaterium WSH-002 strains are available at NCBI data bank. However, was not found any sequence of the phytase in these sequences. Therefore, phytase sequences of different Bacillus species were aligned, and two primer pair with one degenerate was prepared. In PCR (Polymerase Chain Reaction) result, it was realized amplification just using the degenerate primer pairs, and three bands (~900 bp, ~500 bp) were observed. The amplified DNA fragments were transferred into pGEM®-T Easy vector by ligation. Recombinant plasmids were transformed into E.coli DH5α strain, and plasmids of pure E. coli DH5α strains containing the recombinant plasmid was isolated. For control of cloning, the recombinant plasmid was cut with EcoRI restriction endonuclease enzyme, and bands of the expected size were observed in the agarose gel profile. The isolated plasmids were sequenced using T7 primer and the ABI PRISM-310 Genetic Analyzer apparatus. Literature analysis of the sequence determined using the NCBI data bank has been made. According to the results, the nucleotide sequence of all three DNA fragments was determined not show sequence similarity with phytase enzyme. So with, phytase gene as a result of amplification with degenerate primers was not amplified. However, these new isolate is important for literature. This newly isolated Bacillus megaterium sp. EBD 9-1 strain may be great potential for phytase production at industrial scale.
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Keywords
Bacillus, İzolasyon, Fitaz, Klonlama, Bacillus, Isolation, Phytase, Cloning
Citation
Zeren, B. (2015). Topraktan izole edilen Bacillus megaterium EBD 9-1 suşundan fitaz geninin E.coli'de klonlanma çalışmaları. Yayınlanmamış yüksek lisans tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.