PCR amplification and cloning of six target genes from Burkholderia pseudomallei

Burkholderia pseudomallei (B. pseudomallei) is an aerobic Gram negative bacteria and a causative agent of melioidosis, a disease endemic in several regions worldwide including Southeast Asia and Northern Australia. Melioidosis can be fatal in human, where it causes fever and is commonly present...

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Main Authors:	Arifah, Adini Qisthi, Mohamad Nadhan, Hani Nuramalina, Ismail, Shuhaib Ar Rumy, Raih, Mohd Firdaus, Muhamad Bunnori, Noraslinda, Mohamed Rehan, Aisyah
Format:	Conference or Workshop Item
Language:	English
Published:	2016
Subjects:	QR Microbiology
Online Access:	http://irep.iium.edu.my/53576/ http://irep.iium.edu.my/53576/ http://irep.iium.edu.my/53576/13/53576.pdf

id	iium-53576
recordtype	eprints
spelling	iium-535762016-12-28T01:22:17Z http://irep.iium.edu.my/53576/ PCR amplification and cloning of six target genes from Burkholderia pseudomallei Arifah, Adini Qisthi Mohamad Nadhan, Hani Nuramalina Ismail, Shuhaib Ar Rumy Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah QR Microbiology Burkholderia pseudomallei (B. pseudomallei) is an aerobic Gram negative bacteria and a causative agent of melioidosis, a disease endemic in several regions worldwide including Southeast Asia and Northern Australia. Melioidosis can be fatal in human, where it causes fever and is commonly present with pneumonia, with or without septicaemia. Treating melioidosis is a challenge, due to its intrinsic resistance to many antibiotics, and occurrence of latent infection. This study is part of a larger project to purify and characterise target proteins from B. pseudomallei to gain fundamental knowledge on how this bacterium behaves, and thus providing us strategies to combat them. In this report, six target genes predicted to be essential by Transposon-Directed Insertion site Sequencing (TraDIS) technique were selected for PCR amplification and cloning trials. Out of the six target genes, three genes encode for hypothetical proteins (BPSL0084, BPSL0086, and BPSL1060), two genes encode for putative annotated protein (BPSL0398 and BPSL1192) and one gene (BPSL1549) encodes for B. pseudomallei lethal factor 1. Genomic DNA of B. pseudomallei strain D286 and K96243 is obtained from School of Biosciences & Biotechnology, UKM. All six target genes were successfully amplified. Sufficient concentration of purified attBPCR products for all target genes was obtained after nested PCR. This is then utilized in the BP recombination reactions to create the entry clones. The entry clones were transformed into Escherichia coli (E. coli) DH5α™ competent cells and plated onto LB agar plate supplemented with 50 µg/mL kanamycin. The positive clones were screened using colony PCR and BsrGI restriction enzyme digestion. At the time of writing, three target genes were successfully cloned and validated by sequencing. In future projects, the entry clones can then be easily transferred into various expression vectors to be expressed and purified for diverse functional protein studies. 2016-12 Conference or Workshop Item NonPeerReviewed application/pdf en http://irep.iium.edu.my/53576/13/53576.pdf Arifah, Adini Qisthi and Mohamad Nadhan, Hani Nuramalina and Ismail, Shuhaib Ar Rumy and Raih, Mohd Firdaus and Muhamad Bunnori, Noraslinda and Mohamed Rehan, Aisyah (2016) PCR amplification and cloning of six target genes from Burkholderia pseudomallei. In: 33rd Symposium of the Malaysian Society for Microbiology 2016 : Translating Microbiology to Bioeconomy, 14th-17th December 2016, Melaka. (Unpublished) http://www.mymicro.org/msm2016.php
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 Arifah, Adini Qisthi Mohamad Nadhan, Hani Nuramalina Ismail, Shuhaib Ar Rumy Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah PCR amplification and cloning of six target genes from Burkholderia pseudomallei
description	Burkholderia pseudomallei (B. pseudomallei) is an aerobic Gram negative bacteria and a causative agent of melioidosis, a disease endemic in several regions worldwide including Southeast Asia and Northern Australia. Melioidosis can be fatal in human, where it causes fever and is commonly present with pneumonia, with or without septicaemia. Treating melioidosis is a challenge, due to its intrinsic resistance to many antibiotics, and occurrence of latent infection. This study is part of a larger project to purify and characterise target proteins from B. pseudomallei to gain fundamental knowledge on how this bacterium behaves, and thus providing us strategies to combat them. In this report, six target genes predicted to be essential by Transposon-Directed Insertion site Sequencing (TraDIS) technique were selected for PCR amplification and cloning trials. Out of the six target genes, three genes encode for hypothetical proteins (BPSL0084, BPSL0086, and BPSL1060), two genes encode for putative annotated protein (BPSL0398 and BPSL1192) and one gene (BPSL1549) encodes for B. pseudomallei lethal factor 1. Genomic DNA of B. pseudomallei strain D286 and K96243 is obtained from School of Biosciences & Biotechnology, UKM. All six target genes were successfully amplified. Sufficient concentration of purified attBPCR products for all target genes was obtained after nested PCR. This is then utilized in the BP recombination reactions to create the entry clones. The entry clones were transformed into Escherichia coli (E. coli) DH5α™ competent cells and plated onto LB agar plate supplemented with 50 µg/mL kanamycin. The positive clones were screened using colony PCR and BsrGI restriction enzyme digestion. At the time of writing, three target genes were successfully cloned and validated by sequencing. In future projects, the entry clones can then be easily transferred into various expression vectors to be expressed and purified for diverse functional protein studies.
format	Conference or Workshop Item
author	Arifah, Adini Qisthi Mohamad Nadhan, Hani Nuramalina Ismail, Shuhaib Ar Rumy Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah
author_facet	Arifah, Adini Qisthi Mohamad Nadhan, Hani Nuramalina Ismail, Shuhaib Ar Rumy Raih, Mohd Firdaus Muhamad Bunnori, Noraslinda Mohamed Rehan, Aisyah
author_sort	Arifah, Adini Qisthi
title	PCR amplification and cloning of six target genes from Burkholderia pseudomallei
title_short	PCR amplification and cloning of six target genes from Burkholderia pseudomallei
title_full	PCR amplification and cloning of six target genes from Burkholderia pseudomallei
title_fullStr	PCR amplification and cloning of six target genes from Burkholderia pseudomallei
title_full_unstemmed	PCR amplification and cloning of six target genes from Burkholderia pseudomallei
title_sort	pcr amplification and cloning of six target genes from burkholderia pseudomallei
publishDate	2016
url	http://irep.iium.edu.my/53576/ http://irep.iium.edu.my/53576/ http://irep.iium.edu.my/53576/13/53576.pdf
first_indexed	2023-09-18T21:15:46Z
last_indexed	2023-09-18T21:15:46Z
_version_	1777411551913836544

PCR amplification and cloning of six target genes from Burkholderia pseudomallei

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