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Ganesh, Irisappan,Tran, Buu Minh,Kim, Yonghee,Kim, Jaewon,Cheng, Hua,Lee, Nae Yoon,Park, Sungsu Springer-Verlag 2016 Biomedical microdevices Vol.18 No.6
<P>There is growing interest in rapid microbial preconcentration methods to lower the detection limit of bacterial pathogens of low abundance in samples. Here, we report an integrated microfluidic PCR system that enables bacterial cells of interest in samples to be concentrated prior to PCR. It consists of two major compartments: a preconcentration chamber for the immunomagnetic separation of bacterial cells, and a PCR chamber for the DNA amplification of the concentrated cells. We demonstrate the feasibility of the system for the detection of microbial pathogens by preconcentrating the human pathogen Escherichia coli O157: H7, and also amplifying its DNA. The detection limit of E. coli O157: H7 in the PCR system is 1 x 103 CFU (colony forming unit)/mL. Onchip processing steps, including preconcentration and PCR steps, take less than two hours. Our system can serve as a rapid, specific, and quantitative platform for the detection of microbial pathogens in samples of large volume.</P>
Irisappan Ganesh,홍순호,SAMBANDAM RAVIKUMAR,박시재,Seung Hwan Lee 한국화학공학회 2013 Korean Journal of Chemical Engineering Vol.30 No.7
The malate-pyruvate conversion pathway is catalyzed by two malic enzyme isomers, MaeA and MaeB. qRT-PCR was carried out under malate and pyruvate supplemented conditions to understand the dynamics of maeA and maeB gene expression. maeA expression was elevated by malate, and maeB expression was inhibited by levels of both malate and pyruvate higher than 0.1 mM. Green fluorescent protein (GFP) reporter plasmids were also constructed by integration of the maeA/maeB promoter with the gfp gene. We showed that maeA driven GFP expression was positively and negatively correlated with extracellular malate and pyruvate induction. In contrast, no significant changes in maeB driven GFP expression were observed under both malate and pyruvate supplemented conditions.
Irisappan Ganesh,SAMBANDAM RAVIKUMAR,홍순호 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.4
Currently, a variety of feedstock is utilized by metabolically engineered bacteria for the production of bioenergy and biochemicals. Recent studies have shown that glycerol can be used as an alternative feedstock for glucose, considering its higher availability, lower price, and high degree of reduction. Hence, this review focuses on recent developments in the bioconversion of glycerol to bioenergy (ethanol and hydrogen) and biochemicals (1,3-propanediol, 1,2-propanediol, 3-hydroxypropionic acid,succinic acid, lactic acid, polyhydroxyalkanoates and Lphenyl alanine) using metabolically engineered Escherichia coli.
Irisappan Ganesh,Tae-Wan Kim,Jeong-Geol Na,엄경태,Soon Ho Hong 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.1
The Two-component Regulatory System (TCS) is the primary mode that bacteria use to continuously sense the environment. A TCS is comprised of a periplasmic sensor Histidine kinase (HK) domain and a cytoplasmic Response regulator (RR) domain. The HK domain phosphorylates the RR domain to activate the effector gene expression. Utilizing a rational approach, the sensor HK was genetically engineered in Escherichia coli to create chimeric HK, by a rewiring or domain swapping strategy. Apart from the wild-type characteristics, chimeric HK imparts novel or the desired characteristics and ability to genetically engineered E. coli for its adaptation and survival. This review focuses on the design, potential applications, and future perspectives of chimeric HKs used as high throughput screening biosensors of various compounds.
( Irisappan Ganesh ),( Selvamani Vidhya ),( Gyeong Tae Eom ),( Soon Ho Hong ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.6
Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.
Irisappan Ganesh,Murali Kannan Maruthamuthu,유익근,홍순호 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.5
A positive feedback loop was introduced to modify the dynamic behavior of fumarate sensing DcuSZ chimera TCS. To construct the positive feedback loop, the ompR gene was cloned downstream of the ompC promoter. The ompC promoter induced the expression of OmpR, which in turn induced the expression of the ompC promoter. Through the introduction of this positive feedback loop, the transcriptional expression levels of ompC increased 2.6-fold. When GFP was used as a reporter protein, a 64% increase in fluorescence level was observed. These results suggest that sensitivity of the TCS based fumarate sensing system can be engineered through the introduction of a positive feedback loop.
SAMBANDAM RAVIKUMAR,Irisappan Ganesh,Murali Kannan Maruthamuthu,홍순호 한국화학공학회 2015 Korean Journal of Chemical Engineering Vol.32 No.10
In an attempt to create an acidic amino acid-sensing Escherichia coli, a chimeric sensor kinase (SK)-based biosensor was constructed using Pseudomonas putida AauS. AauS is a sensor kinase that ultimately controls expression of the aau gene through its cognate response regulator AauR, and is found only in P. putida KT2440. The AauZ chimera SK was constructed by integration of the sensing domain of AauS with the catalytic domain of EnvZ to control the expression of the ompC gene in response to acidic amino acids. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as the concentration of acidic amino acids increased. These data suggest that AauS-based recombinant E. coli can be used as a bacterial biosensor of acidic amino acids. By employing the chimeric SK strategy, various bacteria biosensors for use in the development of biochemicalproducing recombinant microorganisms can be constructed.
( Vidhya Selvamani ),( Irisappan Ganesh ),( Sowon Chae ),( Murali Kannan Maruthamuthu ),( Soon Ho Hong ) 한국미생물 · 생명공학회 2020 한국미생물·생명공학회지 Vol.48 No.1
Five genes (mxbDM, mxcQE and mxaB) are responsible for the transcription of methanol oxidation genes in Methylobacterium strains. Among these, MxbDM and MxcQE constitute the two-component system (TCS) regulating methanol metabolism. In this study, we integrated the methanol-sensing domain of MxbD and MxcQ with the EnvZ/OmpR from Escherichia coli. The domain-swapping strategy resulted in chimeric histidine kinases (HK’s) MxbDZ and MxcQZ AM1 containing recombinant E. coli. Real-time quantitative PCR was used to monitor OmpC expression mediated by the chimeric HK and response regulator (RR) OmpR. Further, an ompC promoter based fluorescent biosensor for sensing methanol was developed. GFP fluorescence was studied both qualitatively and quantitatively in response to environmental methanol. GFP measurement also confirmed ompC expression. Maximum fluorescence was observed at 0.05% methanol and 0.01% methanol using MxbDZ and MxcQZ AM1, respectively. Thus the chimeric HK containing E. coli were found to be highly sensitive to methanol, resulting in a rapid response making them an ideal sensor.