Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique

Melioidosis is an infectious disease caused by a bacterium called Burkholderia pseudomallei found in contaminated water and soil. B. pseudomallei is naturally resistant to many commonly used antibiotics and thus current research efforts focus on prevention of disease and finding ways to reduce mo...

Full description

Bibliographic Details
Main Authors:	Drahaman, Siti Marhamah, Raih, Mohd Firdaus, Muhamad Bunnori, Noraslinda, Mohamed Rehan, Aisyah
Format:	Conference or Workshop Item
Language:	English
Published:	Centre og Graduate Studies, Kolej Universiti Islam Selangor (KUIS) 2016
Subjects:	QR Microbiology
Online Access:	http://irep.iium.edu.my/58209/ http://irep.iium.edu.my/58209/ http://irep.iium.edu.my/58209/1/58209_CLONING%20AND%20EXPRESSION%20OF%20HYPOTHETICAL.pdf

id	iium-58209
recordtype	eprints
spelling	iium-582092017-09-05T07:58:48Z http://irep.iium.edu.my/58209/ Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique Drahaman, Siti Marhamah Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah QR Microbiology Melioidosis is an infectious disease caused by a bacterium called Burkholderia pseudomallei found in contaminated water and soil. B. pseudomallei is naturally resistant to many commonly used antibiotics and thus current research efforts focus on prevention of disease and finding ways to reduce mortality. Identification of B. pseudomallei essential genes and its products may represent excellent targets for development of novel antimicrobial drugs. In this study, primers were designed for the PCR amplification of five target genes selected based on bioinformatics analysis from transposon-directed insertion site sequencing (TraDIS) library which compiles hypothetical proteins. Successfully amplified target genes were cloned into Gateway™ plasmid before being transformed into E. coli host. Expression trials of the target protein were performed for affinity tag protein purification. The presence of expressed soluble or non-soluble proteins were observed using SDS-PAGE electrophoresis. From this study, five selected target genes were successfully amplified using two-step PCR. One target gene, BPSL 2774 was successfully cloned into Gateway™ pDEST15 (GST-tagged) and purified using glutathione affinity protein purification kit. Mass spectrometry result has confirmed the presence of expressed and partially soluble GST-tagged protein of BPSL 2774. Using BLASTp search to PDB database and I-TASSER structure and function prediction softwares, BPSL 2774 is shown to have conserved domains of Glycosyltransferase GTB type superfamily and predicted to function as a glycosyltransferase. This enzyme is important in cell wall biosynthesis and transfer of sugar. This work provides the foundation for further investigation into the function of hypothetical protein BPSL2774, its possible role as a glycosyltransferase and as a potential virulence factor for B. pseudomallei. Centre og Graduate Studies, Kolej Universiti Islam Selangor (KUIS) 2016-12-02 Conference or Workshop Item PeerReviewed application/pdf en http://irep.iium.edu.my/58209/1/58209_CLONING%20AND%20EXPRESSION%20OF%20HYPOTHETICAL.pdf Drahaman, Siti Marhamah and Raih, Mohd Firdaus and Muhamad Bunnori, Noraslinda and Mohamed Rehan, Aisyah (2016) Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique. In: 3rd International Conference on Postgraduate Research (ICPR2016) – “Connecting Minds To The World”, 1st-2nd Dec. 2016, Bukit Jambul, Penang. http://conference.kuis.edu.my/icpr16/FRONT%20PAGE%20E%20PROS.html
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 Drahaman, Siti Marhamah Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique
description	Melioidosis is an infectious disease caused by a bacterium called Burkholderia pseudomallei found in contaminated water and soil. B. pseudomallei is naturally resistant to many commonly used antibiotics and thus current research efforts focus on prevention of disease and finding ways to reduce mortality. Identification of B. pseudomallei essential genes and its products may represent excellent targets for development of novel antimicrobial drugs. In this study, primers were designed for the PCR amplification of five target genes selected based on bioinformatics analysis from transposon-directed insertion site sequencing (TraDIS) library which compiles hypothetical proteins. Successfully amplified target genes were cloned into Gateway™ plasmid before being transformed into E. coli host. Expression trials of the target protein were performed for affinity tag protein purification. The presence of expressed soluble or non-soluble proteins were observed using SDS-PAGE electrophoresis. From this study, five selected target genes were successfully amplified using two-step PCR. One target gene, BPSL 2774 was successfully cloned into Gateway™ pDEST15 (GST-tagged) and purified using glutathione affinity protein purification kit. Mass spectrometry result has confirmed the presence of expressed and partially soluble GST-tagged protein of BPSL 2774. Using BLASTp search to PDB database and I-TASSER structure and function prediction softwares, BPSL 2774 is shown to have conserved domains of Glycosyltransferase GTB type superfamily and predicted to function as a glycosyltransferase. This enzyme is important in cell wall biosynthesis and transfer of sugar. This work provides the foundation for further investigation into the function of hypothetical protein BPSL2774, its possible role as a glycosyltransferase and as a potential virulence factor for B. pseudomallei.
format	Conference or Workshop Item
author	Drahaman, Siti Marhamah Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah
author_facet	Drahaman, Siti Marhamah Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah
author_sort	Drahaman, Siti Marhamah
title	Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique
title_short	Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique
title_full	Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique
title_fullStr	Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique
title_full_unstemmed	Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique
title_sort	cloning and expression of hypothetical protein targets in burkholderia pseudomallei by transposon-directed insertion site sequencing (tradis) technique
publisher	Centre og Graduate Studies, Kolej Universiti Islam Selangor (KUIS)
publishDate	2016
url	http://irep.iium.edu.my/58209/ http://irep.iium.edu.my/58209/ http://irep.iium.edu.my/58209/1/58209_CLONING%20AND%20EXPRESSION%20OF%20HYPOTHETICAL.pdf
first_indexed	2023-09-18T21:22:17Z
last_indexed	2023-09-18T21:22:17Z
_version_	1777411962018201600

Cloning and expression of hypothetical protein targets in burkholderia pseudomallei by Transposon-Directed Insertion Site Sequencing (TraDIS) technique

Similar Items