Page 55 - GPD-2-4

Gene & Protein in Disease

ORIGINAL RESEARCH ARTICLE
K fragment as a polymerase chain

reaction-based vector for antibiotic resistance
gene hunting

1,2
1
Hanife Salih Doğan *, Erman Oryaşın , and Bülent Bozdoğan 1,3
1 REDPROM Centre (Recombinant DNA and Recombinant Protein Research Centre), Aydın
Adnan Menderes University, Aydın, Turkey
2 Aydın Vocational School of Health Services, Aydın Adnan Menderes University, Aydın, Turkey
3 Department of Medical Microbiology, Medical Faculty, Aydın Adnan Menderes University,
Aydın, Turkey

Abstract
Antimicrobial resistance (AMR) has emerged as an escalating health issue in the
global public health arena. To evaluate and predict AMR, it is of utmost importance to
identify and characterize both the known and unknown genes responsible for AMR.
While known genes can be readily detected, identifying unknown genes present a
challenge. In this study, we developed the plasmid K fragment (pKF) by modifying
the pUC19 vector, specifically by removing the multiple cloning site and introducing
a Prom-RBS sequence. pKF was used for amplification of K fragment that contains
a ribosomal binding site (RBS), and promoter at both ends 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. The cloning experiment
*Corresponding author: demonstrated the usability of this newly developed cloning method with K fragment.
Hanife Salih Doğan
(hanifesalih94@gmail.com) K fragment is an innovative vector that can be easily obtained through amplification by
polymerase chain reaction and lacks antibiotic resistance markers. This novel approach
Citation: Doğan HS, Oryaşın E,
and Bozdoğan B, et al., 2023, K is convenient to use since it allows cloning of resistance genes at all orientations and
fragment as a polymerase chain this flexibility can be maneuvered by changing restriction enzymes for primers and
reaction-based vector for antibiotic fragments. With these distinctive features, this vector stands out to be a versatile tool
resistance gene hunting. Gene for cloning both known and unknown resistance genes, and the improved method
Protein Dis, 2(4): 1674.
https://doi.org/10.36922/gpd.1674 with K fragment enables the microbiological and molecular characterization of cloned
genes. K fragment can be utilized for cloning of resistance genes in bacteria originated
Received: August 24, 2023
Accepted: November 20, 2023 in different environments without having to perform bacterial culture. We believe
Published Online: December 7, 2023 that the convenience brought by this technique could lend itself efficient in the battle
Copyright: © 2023 Author(s). against the growing AMR crisis through pre-emptive identification of resistance genes.
This is an Open Access article
distributed under the terms of the
Creative Commons Attribution Keywords: Antimicrobial resistance; Resistance gene; Cloning vector; Cloning method;
License, permitting distribution, K fragment
and reproduction in any medium,
provided the original work is
properly cited.
Publisher’s Note: AccScience
Publishing remains neutral with 1. Introduction
regard to jurisdictional claims in
published maps and institutional The World Health Organization has declared antimicrobial resistance (AMR) as one of
affiliations. the most important health problems in the world and more than 750,000 people die due

Volume 2 Issue 4 (2023) 1 https://doi.org/10.36922/gpd.1674