Year: 2022
Type: preprint
Abstract: Antimicrobial resistance (AMR) is an important health issue in the world. To evaluate and predict, known and unknown resistance genes that are responsible for AMR should be identified and characterized. Unlike known genes, unknown genes are difficult to detect. In this study, we generated the plasmid of K fragment (pKF) from pUC19 vector by removing multiple cloning site (MCS) and adding Prom-RBS sequence. pKF was used for amplification of K fragment that contains RBS and promoter at both directions and plasmid origin of replication. The functionality of added Prom-RBS sequence and K fragment as a cloning vector was tested by cloning chloramphenicol resistance gene amplicon and erythromycin resistance gene from genomic DNA, respectively. Cloning of them confirmed the usability of this newly developed cloning method with K fragment. K fragment is a PCR based and antibiotic resistance gene-free cloning vector. It allows cloning of resistance genes at all orientations just by changing restriction enzymes for primers and fragmentation. This method enables the microbiological and molecular characterization of cloned genes. We expect the use of K fragment for cloning of resistance genes from different environments without cultivation. It will contribute to fighting AMR by identification of resistance genes before their dissemination.
Source: Research Square
Authors: Hanife Salih, Erman Oryaşın, Bülent Bozdoğan
Institution: Adnan Menderes University
Language: English
Subfield: Molecular Medicine
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4282011348