• Genomics & Informatics
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• v.2 no.4
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• pp.174-179
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• 2004

We developed various plasmid cloning vectors that are useful in the construction of genomic and shotgun libraries. Two medium copy vectors, pCUGlblu21 (pCb21) and pAGlblu21 (pAb21), which are resistant to kanamycin ($Km^R$) and chloramphenicol ($Cam^R$), respectively, are useful for cloning DNA inserts ranging from 5kb to 15kb. Two high copy vectors, pCUGlblu31 (pCb31) and pAGlblu31 (pAb31), containing $Km^R$ and $Cam^R$, respectively, are useful for DNA inserts less than 5kb. These vectors are well adapted for large-scale genome sequencing projects by providing choice of copy number and selectable marker. The small vector size is another advantage of these vectors. All vectors contain lacZa including multicloning sites that originated from pBluscriptllsk- for easy cloning and sequencing. Two medium copy vectors contain unique and rare cutting Swal (ATTTAAAT) restriction enzyme sites for easy determination of insert size. We developed two combined vectors, pC21A31 and pC31A21, which are combinations of (pCb21 + pAb31) and (pCb31 + pAb21), respectively. These two vectors provide four choices of vectors such as $Km^R$ and medium, $Cam^R$ and high, $Cam^R$ and medium, and $Km^R$ and high copy vectors by restriction enzyme cutting, dephosphorylation, and gel purification. These vectors were successfully applied to high throughput shotgun sequencing of rice, tomato, and brassica BAC clones. With an example of extremely biased hydro sheared 3 kb shotgun library of a tomato BAC clone, which is originated from cytogenetically defined peri-centromeric region, we suggest the utility of an additional 10 kb library for sequence assembly of the difficult-to-assemble BAC clone.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.321-324
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• 2001

Two recombinant Escherichia coli strains, GCSC6576 harboring a plasmid pSYL107 containing the Ralstonia eutropha polyhyclroxyalkanoate (PHA) biosynthesis genes and a fadR atoC mutant LS5218 harboring a plasmid pJC4 containing the Alcaligenes latus PHA biosynthesis genes were compared for their ability to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from whey as the sale carbon source. With the pH-stat fed-batch culture of E. coli LS5218, 、 ,ve obtained a cell concentration, a P(3HB-co-3HV) concentration. a P(3HB-co-3HV) content, and a 3HV fraction of 31.8 g/L, 10.6 g/L, 33.4 wt%. and 6.26 mol%, respectively at 39 h.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.388-398
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• 1998

A bacterium which is resistant to both mercury and cadmium, and also capable of utilizing phenol as a carbon and energy source, was isolated from the Kumho River sediments near Kangchang Bridge, Taegu, Korea. The isolate was labeled Pseudomonas sp. KM10 and characterized. The bacteria grew in 4 mM $CdCl_2$and in $70{\mu}M$ $HgCl_2$. The bacteria efficiently removed over 90% of 1 g/l phenol within 30 h. In the presence of 1.250 g/l phenol, the growth of the microorganism was slightly retarded and the microorganism could not tolerate 1.5 g/l phenol. Curing of plasmid from the bacteria was carried out to generate a plasmidless strain. Subsequent experiments localized the genes for phenol degradation in plasmid and the genes for mercury resistance and cadmium resistance on the chromosome. Dot hybridization and Southern hybridization under low stringent conditions were performed to identify the DNA homology. These results showed significant homologies between the some sequence of the chromosome of Pseudomonas sp. KM10 and merR of Shigella flexneri R 100, and between the some sequence of the chromosome of Pseudomonas sp. KM10 and cadA of Staphylococcus aureus pI258. The mechanism of cadmium resistance was efflux, similar to that of S. aureus pI258 cadA, and the mechanism of mercury resistance was volatilization, similar to that of S. flexneri R100 mer.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.275-281
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• 1995

From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and pYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37$\circ$C. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase- negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS- PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.1-11
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• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.

• Journal of Microbiology
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• v.41 no.2
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• pp.89-94
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• 2003

Bacterial strain No. P08 isolated from wastewater at the Cheongju industrial complex was found to be capable of degrading 4-chlorobenzoate under aerobic condition. P08 was identified as Comamonas sp. from its cellular fatty acid composition and 16S rDNA sequence. The fcb genes, responsible for the hydrolytic dechlorination of 4-chlorobenzoate, were cloned from the plasmid pJJl of Comamonas sp. P08. The fcb gene cluster of comamonas sp. PO8 was organized in the order fcbB-fcbA-fcbTl-fcbT2-fcbT3-fcbC. This organization of the fcb genes was very similar to that of the fcb genes carried on the chromosomal DNA of pseudomonas sp. DJ-12. However, it differed from the fcbA-fcbB -fcbC ordering of Arthrobacter sp. SU. The nucleotide sequences of the fcbABC genes of strain P08 showed 98% and 53% identities to those of Pseudomonas sp. DJ-12 and Arthrobacter sp. SU, respectively. This suggests that the fcb genes might have been derived from Pseudomonas sp. DJ-12 to form plasmid pJSl in Comamonas sp. P08, or that the fcb genes in strain DJ-12 were transposed from Comamonas sp. P08 plasmid.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.40-45
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• 1987

The gene for the Bdi I modification enzyme, which is one of Bdi I restriction-modification system, fromBrevibacterium divaricatum FERM 5948 was cloned and expressed in E. coli. For cloning of the Bdi I methylase gene, we have initially used three cloning site(EcoRI, BamHI and Sal I) of plasmid vector pBR 322 and adopted the retransformation method after Bdi I restriction endonuclease cleavage. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by Bdi I restriction enzyme, and the recombinant plasmid pBDIM 116 containing 5.6kb EcoRI insery was proved to carry the gene. Crude cell extracts prepared from strains carrying the plasmid pBDIM 116 contained an S-adenosylmethionine-dependent methyltransferase activity specific for the Bdi I recognition site, ATCGAT. The restriction map was constructed with 11 restriction enzyme, and the Bdi I restriction-modification system was also discussed.

• Research in Plant Disease
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• v.29 no.1
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• pp.94-99
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• 2023

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.415-419
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• 2006

The epoxide hydrolase (EH) of Rhodotorula glutinis which has a high enantioselectivity against aromatic epoxide substrates was expressed to high levels in Escherichia coli based on codon usage. We analysed the Preference of codon usage between the yeast, R. glutinis, and bacteria, E. coli. E. coli, Rosetta(DE3)pLysS, harbors pRARE plasmid with tRNA genes for rare-codons was employed as a host strain. The recombinant E. coli expressing R. glutinis EH showed an enhanced enantioselective hydrolysis activity toward racemic styrene oxide. Enantiopure (S)-styrene oxide with a high enantiopurity of 99% ee (enantiomeric excess) was obtained from racemic substrates.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.260-264
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• 1992

Pseudotnorju.c sp. EL-071P degrading 2.4.5-trichlorophe~~oxyi~cetaicci d (2.3.5-T) was resistantto antibiotics: rifampicin. ampicillin. kanamycin and metal ions : Zn" and Cu".The plasmitl related to the degradation of 2.4.5-'r and rifa~npicin resistance was isolatecifrom the strain. Its size was about 40 Kb. As result of transforming the plasmid intoEsch~rirhiti coli MClOhl, it was confirmed that the plasmid ura.; related to 2.4.5-T degradation.The strain coulil grow in the various chlorinated aromatic analogs as the solc carbon source.In the case of chlorophcnols. the chlorinated mono-substituteti phenols were easily dcgradetlin the order ol' ortho-. ~ ~ a r um- ,c ~tu-position.T he 2.3.5-T mctaholism was inhibited by 4-chlorophenol of 2.4.5-7' analog. In non-chlorinateci aromatics. ~ C I I L O ~ I ~ Csa.l icylilte i~ndtoluene were uscd ax the carbon source by the strain and typestrain Acudonlotrtr.\ plrtirltrKCTC 1643 having clegrad;~bility of various aromatics. But naphtalene was usecl only bythe A~urlomonri.\ sp. EL-07 1 P.the A~urlomonri.\ sp. EL-07 1 P.