pRTA, a new vector and TA cloning: A method for characterization of novel antibiotic resistance genes

Antibiotics are among the most widely used drugs. They are used in the treatment of infections as well as in order to accelerate the growth of livestock and plants. Unconscious use of antibiotics, which remain active after excreted from the body, is an important factor in the development of antibiotic resistance. Over time, the frequency and prevalence of strains with multiple antibiotic resistance increases. The identification of the genes that cause antibiotic resistance is important to reveal the importance of the hazard. The main aim of this study is to develop a method that allows cloning of known or unknown genes that cause antibiotic resistance. For this purpose, it is aimed to develop a vector in which the expression can be obtained from both orientations in the multiple cloning region in order to avoid orientation problems in the cloning of antibiotic resistance genes. The backbone of the pRTA vector generated was obtained by inverse PCR using the pUC19 vector. The promoter and RBS sequence designed in this study were placed at the targeted location in the vector backbone.The placement of the promoter and RBS into the vector which has dual promoter was determined by sequence analysis and cloning studies. The vector which carries adenin base at 3' overhang used in the developed cloning method was obtained by amplifiying pRTA vector and it is called AT vector. The total DNA of S. aureus, which is known to have erythromycin resistance, was randomly fragmented with the fragmentase enzyme and fragments of desired sizes were created. After adding thymine base to 3' overhang of fragments, the fragments become complementary to AT vector and fragments and vector were ligated. The ligand was transferred to the E. coli DH10B strain and the colonies were selected on the agar that contains 200 μg/ml erythromycin. The inserts at the transformants were sequenced and it was found that the gene providing resistance was ermC and it was carried on the plasmid. As a result, the vector constructed has been developed with a dual promoter that provides expression of the multiple cloning site and allows for blue-white selection. The AT vector amplified from developed pRTA vektor is more fruitful compared to plasmid isolation. The fragments containing the resistance genes to be cloned were cleaved from random locations with the fragmentase enzyme, thereby reducing the possibility of being cut through the resistance gene. It also increased the cloning success by allowing the resistance gene to produce fragments that can be cloned. In this thesis, a new vector and a new method for cloning known and unknown resistance genes have been developed.