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

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 br...

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Main Authors:	Mohamed Rehan, Aisyah, Enche Othman, Mohamad Izwan, Mohd Amin, Nor Munirah, Shahdan, Intan Azura, Ahmad Yusof @ Hanafi, Hanani
Format:	Conference or Workshop Item
Language:	English
Published:	2017
Subjects:	QR Microbiology
Online Access:	http://irep.iium.edu.my/57963/ http://irep.iium.edu.my/57963/13/57963.pdf

id	iium-57963
recordtype	eprints
spelling	iium-579632017-08-17T07:44:57Z http://irep.iium.edu.my/57963/ PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae Mohamed Rehan, Aisyah Enche Othman, Mohamad Izwan Mohd Amin, Nor Munirah Shahdan, Intan Azura Ahmad Yusof @ Hanafi, Hanani QR Microbiology 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. 2017 Conference or Workshop Item NonPeerReviewed application/pdf en http://irep.iium.edu.my/57963/13/57963.pdf 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)
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
language	English
topic	QR Microbiology
spellingShingle	QR Microbiology Mohamed Rehan, Aisyah Enche Othman, Mohamad Izwan Mohd Amin, Nor Munirah Shahdan, Intan Azura Ahmad Yusof @ Hanafi, Hanani PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae
description	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.
format	Conference or Workshop Item
author	Mohamed Rehan, Aisyah Enche Othman, Mohamad Izwan Mohd Amin, Nor Munirah Shahdan, Intan Azura Ahmad Yusof @ Hanafi, Hanani
author_facet	Mohamed Rehan, Aisyah Enche Othman, Mohamad Izwan Mohd Amin, Nor Munirah Shahdan, Intan Azura Ahmad Yusof @ Hanafi, Hanani
author_sort	Mohamed Rehan, Aisyah
title	PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae
title_short	PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae
title_full	PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae
title_fullStr	PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae
title_full_unstemmed	PCR-based construction and transformation of CodY gene deletion construct in Streptococcus pneumoniae
title_sort	pcr-based construction and transformation of cody gene deletion construct in streptococcus pneumoniae
publishDate	2017
url	http://irep.iium.edu.my/57963/ http://irep.iium.edu.my/57963/13/57963.pdf
first_indexed	2023-09-18T21:21:56Z
last_indexed	2023-09-18T21:21:56Z
_version_	1777411939722330112

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

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