• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.153-157
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• 2006

A soil metagenomic library was constructed using an E. coli-fosmid cloning system with environmental DNAs extracted from Kwangreung forest topsoil. We targeted the genes involved in the biosynthesis of bacterial polyketides. Initially, a total of 36 clone pools (10,800 clones) were explored by the PCR-based method using the metagenomic DNAs from each pool and a degenerate primer set, which has been designed based on the highly conserved regions among ketoacyl synthase (KS) domains in actinomycete type I polyketide synthases (PKS Is). Six clone pools were tentatively selected as positive and further examined through a hybridization-based method for selecting a fosmid clone containing PKS I genes. Colony hybridization was performed against fosmid clones from the 6 positive pools, and finally 4 clones were picked out and confirmed to contain the conserved DNA fragment of KS domains. In this study, we present a simple and feasible sorting method for a desired clone from metagenomic libraries.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.850-855
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• 2013

Finding new antimicrobial activities by functional metagenomics has been shown to depend on the heterologous host used to express the foreign DNA. Therefore, efforts are devoted to developing new tools for constructing metagenomic libraries in shuttle vectors replicatable in phylogenetically distinct hosts. Here we evaluated the use of the Escherichia coli-Bacillus subtilis shuttle vector pHT01 to construct a forest-soil metagenomic library. This library was screened in both hosts for antimicrobial activities against four opportunistic bacteria: Proteus vulgaris, Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. A new antibacterial activity against B. cereus was found upon screening in B. subtilis. The new antimicrobial agent, sensitive to proteinase K, was not active when the corresponding DNA fragment was expressed in E. coli. Our results validate the use of pHT01 as a shuttle vector and B. subtilis as a host to isolate new activities by functional metagenomics.

• Quality Improvement in Health Care
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• v.22 no.2
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• pp.27-40
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• 2016

Purpose: Globally, falls are a major public health problem. The study aimed to evaluate the predictive validity of the Timed Up and Go test (TUGT) as a screening tool for fall risk. Methods: An electronic search was performed Medline, EMBASE, CINAHL, Cochran Library, KoreaMed and the National Digital Science Library and other databases, using the following keywords: 'fall', 'fall risk assessment', 'fall screening', 'mobility scale', and 'risk assessment tool'. The QUADAS-II was applied to assess the internal validity of the diagnostic studies. Thirteen studies were analyzed using meta-analysis with MetaDisc 1.4. Results: The selected 13 studies reporting predictive validity of TUGT of fall risks were meta-analyzed with a sample size of 1004 with high methodological quality. Overall predictive validity of TGUT was as follows. The pooled sensitivity 0.72 (95% confidence interval [CI]: 0.67-0.77), pooled specificity 0.58 (95% CI: 0.54-0.63) and sROC AUC was 0.75 respectively. Heterogeneity among studies was a moderate level in sensitivity. Conclusion: The TGUT's predictive validity for fall risk is at a moderate level. Although there is a limit to interpret the results for heterogeneity between the literature, TGUT is an appropriate tool to apply to all patients at a potential risk of accidental fall in a hospital or long-term care facility.

• BMB Reports
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• v.41 no.11
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• pp.808-813
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• 2008

Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin $H_2$ ($PGH_2$) into prostaglandin $E_2$ ($PGE_2$). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The $K_m$ and $V_{max}$ of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with $I_{C50}$ values of 100 and 200 ${\mu}M$, respectively. As these compounds competitively inhibited the catalysis of $PGH_2$, their binding sites appeared to be located near the $PGH_2$ binding pocket.

• BMB Reports
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• v.44 no.12
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• pp.811-815
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• 2011

Inhalational anthrax is caused by B. anthracis, a virulent sporeforming bacterium which secretes anthrax toxins consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease and is the main determinant in the pathogenesis of anthrax. Here we report the identification of a lead small-molecule inhibitor of anthrax lethal factor by screening an available synthetic small-molecule inhibitor library using fluorescence-based high-throughput screening (HTS) approach. Seven small molecules were found to have inhibitory effect against LF activity, among which SM157 had the highest inhibitory activity. All theses small molecule inhibitors inhibited LF in a noncompetitive inhibition mode. SM157 and SM167 are from the same family, both having an identical group complex, which is predicted to insert into S1' pocket of LF. More potent small-molecule inhibitors could be developed by modifying SM157 based on this identical group complex.

• The Korean Journal of Pesticide Science
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• v.10 no.2
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• pp.149-152
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• 2006

Construction of focused library of 2-benzylimino-1,3-thiazolines 7 through molecular modification of 3-alkyl-2-phenylimino-1,3-thiazolines 1 which showed selective fungicidal activity against rice blast and their fungitoxic activity against 6 kinds of typical plant diseases was described. Fifty four compounds of focused library of 2-benzylimino-1,3-thiazolines 7 were synthesized from the reaction of the corresponding $\gamma$-chloroacetoacetanilides 6 with N-benzyl thioureas 5 by parallel synthetic methodology. As results of fungicidal screening against the typical plant diseases, twenty eight kinds of 7 at 100 ${\mu}g$ $mL^{-1}$ showed the control value over 50% against tomato late blight.

• The Plant Pathology Journal
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• v.31 no.2
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• Entomological Research
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• v.48 no.5
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• pp.384-389
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• 2018

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site-specific recombination of bacteriophage ${\lambda}$. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of $8.4{\times}10^6$ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus-induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi-induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1655-1660
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• 2007

A metagenome is a unique resource to search for novel microbial enzymes from the unculturable microorganisms in soil. A forest soil metagenomic library using a fosmid and soil microbial DNA from Gwangneung forest, Korea, was constructed in Escherichia coli and screened to select lipolytic genes. A total of seven unique lipolytic clones were selected by screening of the 31,000-member forest soil metagenome library based on tributyrin hydrolysis. The ORFs for lipolytic activity were subcloned in a high copy number plasmid by screening the secondary shortgun libraries from the seven clones. Since the lipolytic enzymes were well secreted in E. coli into the culture broth, the lipolytic activity of the subclones was confirmed by the hydrolysis of p-nitrophenyl butyrate using culture supernatant. Deduced amino acid sequence analysis of the identified ORFs for lipolytic activity revealed that 4 genes encode hormone-sensitive lipase (HSL) in lipase family IV. Phylogenetic analysis indicated that 4 proteins were clustered with HSL in the database and other metagenomic HSLs. The other 2 genes and 1 gene encode non-heme peroxidase-like enzymes of lipase family V and a GDSL family esterase/lipase in family II, respectively. The gene for the GDSL enzyme is the first description of the enzyme from metagenomic screening.