Asia Pacific Journal of Molecular Biology and Biotechnology
Background and objectives: One of the most commonly employed bacterial hosts for the synthesis of... more Background and objectives: One of the most commonly employed bacterial hosts for the synthesis of recombinant proteins is Escherichia coli (E. coli). Choosing an ideal host for the production of the protein of interest is an important step in the large-scale production process. Due to its thermostable characteristic, Taq Pol I, which was first isolated from the bacterium Thermus aquaticus (Taq), is now a typical enzyme found in many laboratories. This study aimed to identify the ideal host for large-scale production of Taq Pol I and purify the enzyme under a simple and rapid purification method. Methods: Taq Pol I gene in pSE420 plasmid was overexpressed in E. coli strains DH5α, TOP10 and BL21(DE3) pLysS. The enzyme was purified using Pluthero’s method and dialyzed using Amicon® Ultra-4 Centrifugal Filter. Results: The host strain E. coli TOP10 produced the highest amounts of Taq Pol I, followed by DH5α and BL21(DE3) pLysS. An estimated 4.5 U/μL of Taq Pol I was produced from a 200 ...
Leptospirosis is an emerging zoonotic disease caused by bacterial species of the genus Leptospira... more Leptospirosis is an emerging zoonotic disease caused by bacterial species of the genus Leptospira. However, the regulatory mechanisms and pathways underlying the adaptation of pathogenic and non-pathogenic Leptospira spp. in different environmental conditions remain elusive. Leptospira biflexa is a non-pathogenic species of Leptospira that lives exclusively in a natural environment. It is an ideal model not only for exploring molecular mechanisms underlying the environmental survival of Leptospira species but also for identifying virulence factors unique to Leptospira’s pathogenic species. In this study, we aim to establish the transcription start site (TSS) landscape and the small RNA (sRNA) profile of L. biflexa serovar Patoc grown to exponential and stationary phases via differential RNA-seq (dRNA-seq) and small RNA-seq (sRNA-seq) analyses, respectively. Our dRNA-seq analysis uncovered a total of 2,726 TSSs, which are also used to identify other elements, e.g., promoter and untra...
<p><b>a) <i>S</i>. Typhi.</b> Lane M: 100 bp DNA ladder (Promega), ... more <p><b>a) <i>S</i>. Typhi.</b> Lane M: 100 bp DNA ladder (Promega), Lane 1–6: Serially diluted gDNA from 100 ng to 1 pg, Lane N: negative control <b>b) <i>S</i>. Paratyphi A.</b> Lane M: 100 bp DNA ladder (Promega), Lane 1–6: Serially diluted gDNA from 100 ng to 1 pg, Lane 7: <i>S</i>. Typhi gDNA, Lane N: negative control and <b>c) <i>S</i>. Paratyphi B.</b> Lane M: 100 bp DNA ladder (Promega), Lane 1–7: Serially diluted gDNA from 100 ng to 100 fg, Lane 8: <i>S</i>. Typhi gDNA, Lane N: negative control.</p
<p>(a-c) Schematic representations of <i>S</i>. <i>typhi</i> sRNA g... more <p>(a-c) Schematic representations of <i>S</i>. <i>typhi</i> sRNA genes. Coordinates of depicted genes are based on the completed genome of <i>S</i>. <i>typhi</i> Ty2 (AE014613). Drawings are not according to scale. (d-f) Predicted secondary structures of StyR-3, StyR-36 and StyR-143 sRNAs, respectively using mfold programme.</p
Gregory C. Adam André Adams Siti Aminah Ahmed Gene Ananiev Evan Appleton Marc G. Aucoin Paul Barb... more Gregory C. Adam André Adams Siti Aminah Ahmed Gene Ananiev Evan Appleton Marc G. Aucoin Paul Barber Jason Baron Mike Berke Matthew Boeckeler Jan Willem Borst Latoya Braun John Burczak Gan Chen Shuxun Chen Valerie Chew Edward Chow Eun Ji Chung Silvia Comani Alexandre Costa Cristopher Cowan Michael Daniele Gareth Denyer Xian-Ting Ding Liting Duan Burak Dura Debashis Dutta Richard Eglen Michael Eisenbach Shirzad Fallahi Zhaobao Fan Adam M. Feist Marc Ferrer Paul Freemont Hariprasad Gali Jose L. Garcia-Cordero Joerg Geiger Reza Ghaemi James Gill Alireza Goodarzi Paul Gosnell Catherine Greene Hanspeter Gubler Benhamin Haley Xiaomin He Xiaoming He Nathaniel Hentz Maciej Holowko Stephen Hughes Berthold Huppertz John Janiszewski Steven Jarvis David Jennions Kaveh Jorabchi Samantha Kanza Peter Keizers Doug Kelley David Kelso Pavel Klimes Tadej Kokalj Choong-Weng Lam Hookeun Lee Jong-Hwan Lee Kin Fong Lei Ralf Lenigk Felix Lenk Melanie Leveridge Hui Li Ying-Chi Lin Jonathan Lippy Markus List ...
<p><i>StyR-3</i>, <i>StyR-36</i> and <i>StyR-143</i> sR... more <p><i>StyR-3</i>, <i>StyR-36</i> and <i>StyR-143</i> sRNA gene sequences.</p
Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to... more Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we for-mulated a strategy of detection and differentiation of salmonellosis by a multiplex polymer-ase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecul...
Background: Diagnosis of leptospirosis has traditionally relied on the demonstration of immune-se... more Background: Diagnosis of leptospirosis has traditionally relied on the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular methods including conventional and real-time Polymerase Chain Reaction (PCR) have been developed for the specific detection of pathogenic Leptospira. PCR is a sensitive, specific and rapid technique that has been successfully used to detect several microorganisms, including those of clinical significance. Objective: In this research, we developed of a multiplex PCR (mPCR) for detecting leptospira DNA. The mPCR detects both the 16S rRNA gene as well as major outer membrane lipoprotein LipL32 gene. Representative serovars from 10 species of Leptospira and 23 species of other bacteria species were tested. Results: Positive results were obtained from all leptospiral serovars. The amplification sensitivity for multiplex assay was 21.8 pg and 1 x 103 leptospires/ml. This mPCR has a potential to facil...
Polymerase chain reaction (PCR) is the basis of recombinant and other molecular biological techni... more Polymerase chain reaction (PCR) is the basis of recombinant and other molecular biological techniques. Availability of cheap and robust PCR platforms enables the tests to be performed easily, even in resource constrained settings. Herein we compared the efficacy of a portable thermal cycler ( Palm PCR G1-12 System) for rapid DNA amplification against the standard Peltier-based thermal cycler using plasmid DNA and genomic DNA in single and multiplex PCR experiments. Our study revealed that the Palm PCR G1-12 System could be a portable DNA amplification system to conduct various molecular techniques, especially in places where resources are limited.
World Journal of Microbiology and Biotechnology, 2013
Although the multi-copy and specific element IS6110 provides a good target for the detection of M... more Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify ...
Critical reviews in biochemistry and molecular biology, Jan 24, 2018
Over the past decade, RNA-deep sequencing has uncovered copious non-protein coding RNAs (npcRNAs)... more Over the past decade, RNA-deep sequencing has uncovered copious non-protein coding RNAs (npcRNAs) in bacteria. Many of them are key players in the regulation of gene expression, taking part in various regulatory circuits, such as metabolic responses to different environmental stresses, virulence, antibiotic resistance, and host-pathogen interactions. This has contributed to the high adaptability of bacteria to changing or even hostile environments. Their mechanisms include the regulation of transcriptional termination, modulation of translation, and alteration of messenger RNA (mRNA) stability, as well as protein sequestration. Here, the mechanisms of gene expression by regulatory bacterial npcRNAs are comprehensively reviewed and supplemented with well-characterized examples. This class of molecules and their mechanisms of action might be useful targets for the development of novel antibiotics.
Asia Pacific Journal of Molecular Biology and Biotechnology
Background and objectives: One of the most commonly employed bacterial hosts for the synthesis of... more Background and objectives: One of the most commonly employed bacterial hosts for the synthesis of recombinant proteins is Escherichia coli (E. coli). Choosing an ideal host for the production of the protein of interest is an important step in the large-scale production process. Due to its thermostable characteristic, Taq Pol I, which was first isolated from the bacterium Thermus aquaticus (Taq), is now a typical enzyme found in many laboratories. This study aimed to identify the ideal host for large-scale production of Taq Pol I and purify the enzyme under a simple and rapid purification method. Methods: Taq Pol I gene in pSE420 plasmid was overexpressed in E. coli strains DH5α, TOP10 and BL21(DE3) pLysS. The enzyme was purified using Pluthero’s method and dialyzed using Amicon® Ultra-4 Centrifugal Filter. Results: The host strain E. coli TOP10 produced the highest amounts of Taq Pol I, followed by DH5α and BL21(DE3) pLysS. An estimated 4.5 U/μL of Taq Pol I was produced from a 200 ...
Leptospirosis is an emerging zoonotic disease caused by bacterial species of the genus Leptospira... more Leptospirosis is an emerging zoonotic disease caused by bacterial species of the genus Leptospira. However, the regulatory mechanisms and pathways underlying the adaptation of pathogenic and non-pathogenic Leptospira spp. in different environmental conditions remain elusive. Leptospira biflexa is a non-pathogenic species of Leptospira that lives exclusively in a natural environment. It is an ideal model not only for exploring molecular mechanisms underlying the environmental survival of Leptospira species but also for identifying virulence factors unique to Leptospira’s pathogenic species. In this study, we aim to establish the transcription start site (TSS) landscape and the small RNA (sRNA) profile of L. biflexa serovar Patoc grown to exponential and stationary phases via differential RNA-seq (dRNA-seq) and small RNA-seq (sRNA-seq) analyses, respectively. Our dRNA-seq analysis uncovered a total of 2,726 TSSs, which are also used to identify other elements, e.g., promoter and untra...
<p><b>a) <i>S</i>. Typhi.</b> Lane M: 100 bp DNA ladder (Promega), ... more <p><b>a) <i>S</i>. Typhi.</b> Lane M: 100 bp DNA ladder (Promega), Lane 1–6: Serially diluted gDNA from 100 ng to 1 pg, Lane N: negative control <b>b) <i>S</i>. Paratyphi A.</b> Lane M: 100 bp DNA ladder (Promega), Lane 1–6: Serially diluted gDNA from 100 ng to 1 pg, Lane 7: <i>S</i>. Typhi gDNA, Lane N: negative control and <b>c) <i>S</i>. Paratyphi B.</b> Lane M: 100 bp DNA ladder (Promega), Lane 1–7: Serially diluted gDNA from 100 ng to 100 fg, Lane 8: <i>S</i>. Typhi gDNA, Lane N: negative control.</p
<p>(a-c) Schematic representations of <i>S</i>. <i>typhi</i> sRNA g... more <p>(a-c) Schematic representations of <i>S</i>. <i>typhi</i> sRNA genes. Coordinates of depicted genes are based on the completed genome of <i>S</i>. <i>typhi</i> Ty2 (AE014613). Drawings are not according to scale. (d-f) Predicted secondary structures of StyR-3, StyR-36 and StyR-143 sRNAs, respectively using mfold programme.</p
Gregory C. Adam André Adams Siti Aminah Ahmed Gene Ananiev Evan Appleton Marc G. Aucoin Paul Barb... more Gregory C. Adam André Adams Siti Aminah Ahmed Gene Ananiev Evan Appleton Marc G. Aucoin Paul Barber Jason Baron Mike Berke Matthew Boeckeler Jan Willem Borst Latoya Braun John Burczak Gan Chen Shuxun Chen Valerie Chew Edward Chow Eun Ji Chung Silvia Comani Alexandre Costa Cristopher Cowan Michael Daniele Gareth Denyer Xian-Ting Ding Liting Duan Burak Dura Debashis Dutta Richard Eglen Michael Eisenbach Shirzad Fallahi Zhaobao Fan Adam M. Feist Marc Ferrer Paul Freemont Hariprasad Gali Jose L. Garcia-Cordero Joerg Geiger Reza Ghaemi James Gill Alireza Goodarzi Paul Gosnell Catherine Greene Hanspeter Gubler Benhamin Haley Xiaomin He Xiaoming He Nathaniel Hentz Maciej Holowko Stephen Hughes Berthold Huppertz John Janiszewski Steven Jarvis David Jennions Kaveh Jorabchi Samantha Kanza Peter Keizers Doug Kelley David Kelso Pavel Klimes Tadej Kokalj Choong-Weng Lam Hookeun Lee Jong-Hwan Lee Kin Fong Lei Ralf Lenigk Felix Lenk Melanie Leveridge Hui Li Ying-Chi Lin Jonathan Lippy Markus List ...
<p><i>StyR-3</i>, <i>StyR-36</i> and <i>StyR-143</i> sR... more <p><i>StyR-3</i>, <i>StyR-36</i> and <i>StyR-143</i> sRNA gene sequences.</p
Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to... more Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we for-mulated a strategy of detection and differentiation of salmonellosis by a multiplex polymer-ase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecul...
Background: Diagnosis of leptospirosis has traditionally relied on the demonstration of immune-se... more Background: Diagnosis of leptospirosis has traditionally relied on the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular methods including conventional and real-time Polymerase Chain Reaction (PCR) have been developed for the specific detection of pathogenic Leptospira. PCR is a sensitive, specific and rapid technique that has been successfully used to detect several microorganisms, including those of clinical significance. Objective: In this research, we developed of a multiplex PCR (mPCR) for detecting leptospira DNA. The mPCR detects both the 16S rRNA gene as well as major outer membrane lipoprotein LipL32 gene. Representative serovars from 10 species of Leptospira and 23 species of other bacteria species were tested. Results: Positive results were obtained from all leptospiral serovars. The amplification sensitivity for multiplex assay was 21.8 pg and 1 x 103 leptospires/ml. This mPCR has a potential to facil...
Polymerase chain reaction (PCR) is the basis of recombinant and other molecular biological techni... more Polymerase chain reaction (PCR) is the basis of recombinant and other molecular biological techniques. Availability of cheap and robust PCR platforms enables the tests to be performed easily, even in resource constrained settings. Herein we compared the efficacy of a portable thermal cycler ( Palm PCR G1-12 System) for rapid DNA amplification against the standard Peltier-based thermal cycler using plasmid DNA and genomic DNA in single and multiplex PCR experiments. Our study revealed that the Palm PCR G1-12 System could be a portable DNA amplification system to conduct various molecular techniques, especially in places where resources are limited.
World Journal of Microbiology and Biotechnology, 2013
Although the multi-copy and specific element IS6110 provides a good target for the detection of M... more Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify ...
Critical reviews in biochemistry and molecular biology, Jan 24, 2018
Over the past decade, RNA-deep sequencing has uncovered copious non-protein coding RNAs (npcRNAs)... more Over the past decade, RNA-deep sequencing has uncovered copious non-protein coding RNAs (npcRNAs) in bacteria. Many of them are key players in the regulation of gene expression, taking part in various regulatory circuits, such as metabolic responses to different environmental stresses, virulence, antibiotic resistance, and host-pathogen interactions. This has contributed to the high adaptability of bacteria to changing or even hostile environments. Their mechanisms include the regulation of transcriptional termination, modulation of translation, and alteration of messenger RNA (mRNA) stability, as well as protein sequestration. Here, the mechanisms of gene expression by regulatory bacterial npcRNAs are comprehensively reviewed and supplemented with well-characterized examples. This class of molecules and their mechanisms of action might be useful targets for the development of novel antibiotics.
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Papers by Siti Aminah Ahmed