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2020, Cilt 50, Sayı 1, Sayfa(lar) 027-034
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pADU94, a Cloning and Expression Vector with Chloramphenicol Resistance Marker
Hanife Salih, Erman Oryaşın, Bülent Bozdoğan
Aydın Adnan Menderes Üniversitesi Rekombinant DNA ve Rekombinant Protein Uygulama ve Araştırma Merkezi (REDPROM), Aydın
Keywords: Cloning vector, antibiotic resistance, chloramphenicol, cat

Objective: Nowadays, the vectors with ampicillin and kanamycin resistance genes are widely used to construct a new vector with chloramphenicol -resistance marker that may be used for cloning of the beta- lactam resistance genes.

Method: In this study, pUC19 was used for vector backbone amplification and E. coli DH10B for plasmid transformation. Vector backbone for pADU94 was amplified from pUC19 by inverse PCR by excluding bla gene. Chloramphenicol-resistance gene (cat) was amplified by PCR and ligated to the vector backbone. The constructed pADU94 plasmid was transferred into E. coli DH10B bacteria and the transformants were selected on agar medium containing h 10 μg/ml chloramphenicol. The bla, gfp ve tsst genes were amplified by PCR and cloned into pADU94. The expressions of the cloned genes were tested with the presence of the green fluorescence under the UV for gfp and the penicillin inactivation was demonstrated with modified Hodge test for bla.

Results: The constructed pADU94 vector was transferred into E. coli DH10B and plasmid extraction was done to show presence of pADU94 in transformants for which the MIC value for chloramphenicol was increased from 2 μg/ml to 64 μg/ml. Also, the functionality of the cloned beta lactamase gene was confirmed using modified Hodge test. By cloning tsst gene it has been demonstrated that selection between blue, and white could be realized using f pADU94 plasmid, vector and presence of tsst gene as an insert was verified by plasmid extraction from white colonies. The expression of the cloned gfp gene was verified with observation of green fluorescence under UV light.

Conclusion: According to the obtained results, the constructed pADU94 vector with chloramphenicol resistance marker is a vector that is capable of selecting blue- white colonies and expressing gene. It was shown that pADU94 vector can be used for gene expression and cloning of DNA fragment. The vector has been found to be a suitable vector that can also be used to characterize unknown betalactam resistance genes or to investigate the properties of known beta-lactam resistance genes by way of cloning. With this article a method for construction of vectors with different resistance markers in a laboratory were submitted for the use of rreaders.


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