PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae

Mohamed Rehan, Aisyah and Enche Othman, Mohamad Izwan and Mohd Amin, Nor Munirah and Shahdan, Intan Azura and Ahmad Yusof @ Hanafi, Hanani (2017) PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae. In: Medical Research Symposium 2017, 10th August 2017, Kuantan, Pahang Darul Makmur, Malaysia. (Unpublished)

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Abstract

Introduction: Streptococcus pneumoniae (S. pneumoniae) is a gram-positive diplococci belonging to the genus Streptococcus and it is a well-studied pathogenic bacterium. Pneumococcal diseases such as otitis media, pneumonia, sepsis and meningitis caused by pathogenic strains of S. pneumoniae still brought significant mortality and morbidity rate worldwide. The pathogenicity of S. pneumoniae is exerted by various virulence factors and one of it is the enzyme hyaluronate lyase. Hyaluronate lyase plays major role in invasive capability of S. pneumoniae. Its mechanism of action and crystallographic structure has been determined but its regulatory mechanism is still poorly understood. Drawing connections between nutritional behaviours and invasive property of S. pneumoniae, CodY regulator is hypothesized as a potential hyaluronate lyase regulator. This work is aimed to construct CodY deficient mutant of S. pneumoniae to form foundational work for the study of CodY regulatory effect on hyaluronate lyase. Materials and method: A single gene-deletion method was chosen to create CodY mutant construct containing aphA-3 gene (encoding kanamycin resistance protein) replacement cassette by employing recombinant fusion PCR method. A single band DNA product at the expected size of 2,563 bp after recombinant fusion PCR was visualized on agarose gel electrophoresis. This indicates a high probability that the linear recombinant amplicon has been successfully constructed. The recombinant amplicon was transformed into S. pneumoniae cells of Malaysian local clinical isolate by employing the competence stimulating peptide (CSP-1) to enhance and induce natural competence. Results: No viable CodY mutant was evident post-transformation. The negative transformation results are postulated to be due to the essential nature of CodY regulator. Conclusion: This work can provide basis for recombinant fusion PCR method in designing and constructing single gene deletion construct. However, further works are needed to identify the regulatory agent of hyaluronate lyase.

Item Type:	Conference or Workshop Item (Slide Presentation)
Additional Information:	4469/57963 Aisyah Mohamed Rehan, Mohammad Izwan Enche Othman, Nor Munirah Mohd Amin, Intan Azura Shahdan, Hanani Ahmad Yusof@Hanafi. (2017). PCR-based Construction and Transformation of CodY Gene Deletion Construct in Streptococcus pneumoniae. In abstracts book of Medical Research Symposium 2017, organised by Kulliyyah of Medicine, International Islamic University Malaysia, 10 August 2017, Kulliyyah of Medicine, International Islamic University Malaysia, Indera Mahkota Campus, Jalan Sultan Ahmad Shah, Kuantan, Pahang Darul Makmur, MALAYSIA. Pp. 22. Corresponding author: Mohammad Izwan Enche Othman. (Oral with Abstract ID: 91 - Basic Medical Sciences).
Uncontrolled Keywords:	PCR-based, construction,transformation, CodY gene, Streptococcus pneumoniae
Subjects:	Q Science > QR Microbiology
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button):	Kulliyyah of Allied Health Sciences > Department of Biomedical Science (Effective:1st July 2011) Kulliyyah of Science > Department of Biotechnology
Depositing User:	Mrs Aisyah Mohamed Rehan
Date Deposited:	17 Aug 2017 15:44
Last Modified:	17 Aug 2017 15:44
URI:	http://irep.iium.edu.my/id/eprint/57963

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