Biomedical subjects

Byoung Chan Kim

Publications and source records attributed to Byoung Chan Kim.

7 recordsLinked to original sources

PubMed2005

Multiple and simultaneous detection of specific bacteria in enriched bacterial communities using a DNA microarray chip with randomly generated genomic DNA probes.

A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.

Bacterial Typing Techniques↗

PubMed2004

Development of a DNA microarray chip for the identification of sludge bacteria using an unsequenced random genomic DNA hybridization method.

A tool, based upon the DNA microarray chip, for the identification of specific bacteria from activated sludge, using the hybridization of genomic DNA with random probes, is described. This chip was developed using the genomic DNAs from Gordonia amarae, the natural filamentous actinomycete that causes sludge foaming and bulking, as well as a nonfilamentous floc forming bacterium (Zoogloea ramigera) and the skin pathogen Mycobacterium peregrinum without any sequence information. The sets of target probes on amine-coated glass were made from a genomic library, constructed with PCR products derived from randomly fragmented genomic DNAs extracted from pure cultures of the three strains. Initial hybridization results, when pure cultures were employed, showed the specificity of the probes as well as the resolution of the system, demonstrating the capabilities of this system to identify specific bacterial strains. The microarray was also tested for its ability to distinguish specific bacteria from among mixed bacterial communities, such as in sludge, soil, or spiked genomic DNA samples. The results showed that the probes are specific, with only mild cross-hybridization occurring in a small number of cases. Furthermore, the chip clearly discriminated the presence of all three strains when they were present alone or together within mixed samples. Moreover, using the spot intensity and DNA hybridization kinetics, the starting genomic DNA concentrations could be estimated relatively well, which would make it possible to predict the number of specific bacteria present within the test samples. Therefore, the random genomic hybridization approach, i.e., without any sequence information available for the probes, is a practical protocol for the identification of and screening for specific bacteria within any complex bacterial community from the environmental samples, such as in activated sludge, although the possibility of cross-hybridization may still exist.

Bacteria↗

PubMed2004

Whole-cell-based biosensors for environmental biomonitoring and application.

A variety of whole-cell-based biosensors has been developed using numerous native and recombinant biosensing cells. The use of reporter genes, for example bacterial luciferase and gfp, to monitor gene expression is discussed in terms of each reporters' benefits and disadvantages, including their possible use on-line, their sensitivity, the need for extra substrate, etc. All biosensing cells in use can be classified into two groups in terms of their biosensing mechanisms--constitutive expression and stress- or chemical-specific inducible expression. In this review several examples of each are presented and discussed. The use of recombinant whole-cell biosensors in the field requires three components--biosensing cells, a measurement device, and a signal-transducing apparatus, the last two depending on the first and the final applications of the system. The use of different immobilization techniques in several studies to maintain the cells and their viability is also discussed, in particular their use in the development of both high-throughput and chip-based biosensing systems. Finally the application of whole-cell-based biosensors to different environmental media, such as water, soil, and atmospheric monitoring is discussed; particular attention is given to their use for detection of various stressors, including dioxins, endocrine-disrupting chemicals, and ionizing radiation.

Biosensing Techniques↗

PubMed2003

A bioluminescent sensor for high throughput toxicity classification.

A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon. The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage. In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples. As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased. This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range. In particular, each test requires only 4 h to give clear bioluminescent response signature. Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response. The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.

Biosensing Techniques↗

PubMed2003

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay.

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.

Animals↗

PubMed2003

Monitoring and classification of PAH toxicity using an immobilized bioluminescent bacteria.

An immobilized recombinant bioluminescent Escherichia coli strain, harboring a lac::luxCDABE fused plasmid, which shows lower bioluminescence levels when cellular metabolism is inhibited, was used to monitor the cellular toxicity of polycyclic aromatic hydrocarbons (PAHs). PAHs, classified as pericondensed (PCPAHs) or catacondensed (CCPAHs) according to their molecular structures, were differentiable according to the response of this biosensor. Only CCPAHs were found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output. The induction of cellular toxicity by CCPAHs and PCPAHs was compared with acute toxicity predictions obtained using the quantitative structure-activity relationship (QSAR) model. A good relationship was obtained between the toxicities determined with the bioluminescent response of the immobilized bacterium GC2 and the QSAR model. It was also found that the present study offers a new method of predicting the cellular toxicities of CCPAHs or PCPAHs using this biosensor.

Biosensing Techniques↗

PubMed2002

A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: its cloning, expression, and biochemical properties.

A gene, tayI, encoding a novel subtilisin-like protease, designated thermicin, from the extremely thermophilic bacterium Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was cloned by using a sequence tag containing the consensus sequence of proteases. The gene consisted of 1,239 nucleotides, and the deduced amino acid sequence indicated that it is a preproenzyme with a 311-residue mature protein composed of canonical catalytic residues (Asp29, His64, and Ser252). Thermicin was overproduced in E. coli as a fusion protein with a histidine tag and purified by nickel nitrilotriacetic acid affinity chromatography. Thermicin from E. coli showed maximum proteolytic activity at 92.5 degrees C and pH 9.0, and its half-life was 30 h at 80 degrees C. In order to determine cleavage specificity, thermicin was incubated with insulin beta chain, and the resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The carboxyl group side of the Val12, Leu15,17, Gly23, and Pro28 residues was cleaved. Thermicin is well known to hydrolyze Gly- and Pro-rich collagens. The K (m) and k (cat)/ K (m) values of thermicin for the hydrolysis of the synthetic substrate L-Gly-Pro- p-nitroaniline were 54.16 microM and 142.05 (10(5) s(-1) M(-1)), respectively, at 92.5 degrees C and pH 9.0. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme belongs to a new subgroup with respect to its molecular evolution, when compared with previously characterized subtilisins. This result indicates that thermicin is a novel enzyme different from other thermostable proteases.

Amino Acid Sequence↗