• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2006년도 춘계학술대회 논문집
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• pp.94-95
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• 2006

• 생명과학회지
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• 제21권1호
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• pp.159-164
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• 2011

미생물 세포 바이오센서는 환경오염물질의 모니터링을 위한 좋은 분석도구가 될 수 있다. 이는 리포터유전자들(예로, lux, gfp or lacZ)을 방향족 화합물이나 중금속과 같은 오염물질에 반응하는 유도 조절유전자와 결합하여 만든다. 이러한 유전자 재조합기술을 이용하여 많은 종류의 미생물 바이오센서가 개발되었으며 환경, 의학, 식품, 농업, 및 방위등 다양한 분야에서 활용되고 있다. 또한 바이오센서의 민감도와 검출범위는 조절유전자의 변형을 통해 증가시킬 수있다. 최근에는 미생물 바이오센서 세포를 고효율 검색용 세포 에레이의 칩, 광섬유 등에 고착하여 활용하고 있다. 본 논문은 특이 오염물질의 검출을 위한 유전자 재조합으로 만든 미생물 세포 바이오센서의 현황과 미래에 대해 고찰한다.

• 대한의생명과학회지
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• 제9권4호
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• pp.195-201
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• 2003

Aromatic hydrocarbons are of major concern among genotoxic chemicals due to their toxicity and persistence. Some microorganisms can utilize aromatic hydrocarbons as carbon and energy sources by inducing expression of catabolic operon(s). The XylR regulatory protein activates transcription of the catabolic enzymes to degrade BTEX (benzene, toluene, ethylbenzene, and xylene) from its cognate promoters, Pu and Ps upon exposure of the cells to the aromatic hydrocarbons. The activity of XylR on the promoters was previously monitored using luciferase luc reporter system. The xylR, its promoter Pr and the promoter Po for the phenolic compound catabolic operon were introduced upstream of firefly luciferase luc in the pGL3b vector to generate about 7.1 kb of pXRBTEX. Here E. coli harboring the plasmid was freeze-dried under various conditions to fin,d optimal conditions for storage and transport. The cell viability and luciferase activity were maintained better, when the cells were freeze-dried at -7$0^{\circ}C$ in the addition of the 10% skim milk or 12% sucrose. However, coaddition of protectants such as 10% skim milk plus 10% glucose or 12% sucrose plus 10% glucose, resulted in much better viability and bioluminescence activity compared with the effect of single addition of each protectant. In addition, it was shown that the freeze-dried cells maintained almost intact bioluminescent activities and cell viability for at least 1 week after freeze-drying. This work demonstrated that the properly freeze-dried recombinant bacterial cells could be utilized as a whole-cell biosensor for simple and rapid monitoring of BTEX in the environment.

• Journal of Microbiology and Biotechnology
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• 제33권11호
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• pp.1506-1512
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• 2023

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r² = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

• Molecules and Cells
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• 제39권11호
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• pp.807-813
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• 2016

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of $1.3{\times}10^6CFU/ml$. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate ($5{\times}10^4CFU/ml$). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of $2{\times}10^4CFU/mL$ of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

• Journal of Biosystems Engineering
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• 제39권2호
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• pp.111-114
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• 2014

Purpose: This review aims to introduce aptamers and the methods of its development to improve the sensitivity and selectivity to target bacteria. In this review, we have highlighted current developments and directions in the pathogen detection based on aptamers. Background: Aptamers, the specific nucleic acid sequences, can bind to targets with high affinity and specificity. Some of researches on the use of aptamers for the detection of pathogen have been reported in recent years. Aptamers have more applicability than antibodies for the development of pathogen detection using biosensor; such as easy to synthesis and labeling, lack of immunogenicity, and a low cost of production. However, only few reports on the development and use of aptamers for the detection of pathogen have been published. Review: Aptamers specific to pathogen are obtained by whole-cell systematic evolution of ligands by exponential enrichment (SELEX) process. SELEX process is composed of screening random oligonucleotide bound with target cells, multiple separation and amplification of nucleic acids, final identification of the best sequences. For improving those affinity and selectivity to target bacteria, optimization of multiple separating process to remove unbounded oligonucleotides from aptamer candidates and sorting process by flow cytometry are required.

• Biotechnology and Bioprocess Engineering:BBE
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• 제4권1호
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• pp.59-62
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• 1999

The recombinant bacteria strain DPD2540, containing a fabA::luxCDABE fusion, was used to detect the toxicity of various chemicals in this study. Membrane damaging agents such as phenol, ethanol, and cerulenin induced a rapid bioluminescent response from this strain. Other toxic agents, such as DNA-damaging or oxidative-damaging chemicals, showed a delayed bioluminescent response in which the maximum peak appeared over 150 min after induction. This strain was also tested for measurement of toxicity in field samples such as wastewater and river water effluents.

• KSBB Journal
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• 제32권2호
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• pp.160-167
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• 2017

Coating of individual cells with organic or inorganic materials has drawn a great deal of attention, because it provides the cells with physicochemical durability, which would contribute to the development of bioreactors, biosensor, and lab-on-a-chip, as well as to the fundamental studies in single cell-based biology. Although many strategies have been developed for coating of microbial cells, limited methods are available to coat mammalian cells because most mammalian cells do not have a robust membrane or exoskeleton. Instead, they are enclosed in a lipid bilayer, which is fluidic and vulnerable to changes in its environments. It is more difficult to treat mammalian cells in vitro than microbial cells because the surfaces of mammalian cells are not protected or reinforced by a tough coat. In this work, we report a cytocompatible and degradable nanocoat for mammalian cells. Three types of mammalian cells (HeLa cells, NIH 3T3 fibroblasts, and Jurkat T cells) were individually coated within metal-polyphenol. To maintain the viability of the mammalian cells, we performed the whole processes under strictly physiological culture conditions, and carefully selected nontoxic materials.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1727-1732
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• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.