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 ?g/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 ?g/ml to 64 ?g/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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