• Korean Journal of Microbiology
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• v.28 no.1
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• pp.55-64
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• 1990

Cell volume and DNA contents of the fusants were similar to those of parents. Genetic stability of the fusants was increased when they were cultured on minimal medium (MM) rather than on complete medium (CM), and the fusants were stabilized by subculturing 7 generations each 7 day on MM agar. The finally selected fusants after being cultured for 6 months on CM were stable without segregation. The fusants could also form nuclein and ascospores, and show red and pink colors by the test of TTC colorization. Assimilability and fermentability of carbon sources of the fusants were similarto those of parents. The tolerance of KCl, NaCl, sodium propionate and cycloheximide showed the traits of one strain of parents. When the fusants were cultured for 72 hr and 60 hr in the medium containing 20% glucose and sucrose, respectively, the yield of ethanol for FWKS 260 was reached to 9.6 v/v% and 9.8v/v%, respectively. The sensitive strain Kyokai 7 was found to be killed entirely after cultivation of 48 hr by the killer toxin from the fusants. The recipient S 29 and Kyokai 7 were found to have neither L nor M dsRNA plasmid. However, K 52 and fusants had both L and M dsRNA plasmid of 4.7 kb and 2.5 kb, respectively. The curants treated by heat and cycloheximide did not contain M dsRNA plasmid, but had large amounts of L dsRNA plasmid of those of killer yeasts.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.314-319
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• 2008

Penicillin G amidase (PGA, benzylpenicillinaminohydrolase, EC 3.5.1.11) is industrially important enzyme which converts penicillin G to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The PGA in E. coli ATCC 11105 is secreted into the periplasm after removing signal sequences and becomes heterodimer which composed of two subunits, small subunit (24 kDa) and large subunit (65 kDa). In this study, the PGA gene was obtained from E. coli ATCC 11105 using PCR (polymerase chain reaction) technique. The active PGA was successfully secreated into periplasm in E. coli BL2 1(DE3) harboring pET-pga plasmid. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from $37^{\circ}C$ to $22^{\circ}C$, gave a productivity of 19.6 U/mL with a cell growth of 62 O.D. at 600 nm.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.278-284
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• 1994

Streptomyces spp. purchased from American Type Culture Collection and Institute for Fermentation in Osaka, and donated from Northem Regional Research Laboratory, and those isolated from soil samples were assayed to isolate many plasmids harboring streptomycetes. Among these qrganisms, 5 small size-plasmid carrying organisms SNUS 8810-597A, 8810-600, 8810-754, 8811-344, and 8811-347 were characterized and their plasmids pSJ597, pSJ600, pSJ754, pSJ344, and pSJ347 were isolated in a large scale. The plasmid harboring organisms were sensitive to neomycin, kanamycin, gentamicin, and thiostreptone, but some of them showed weak or strong resistance against streptomycin, chloramphenicol, ampicillin, and tetracycline. It was confirmed that pSJ597 and pSJ600 do not carry antibiotic biosynthetic genes. pSJ600 showed a pock-forming character.

• Journal of Yeungnam Medical Science
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• v.1 no.1
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• pp.41-48
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• 1984

The soil bacterium Agrobacterium tumefaciens is a plant pathogen that cause3 crown gall tumors by infecting the wounded dicotyledonous plants and subsequent integration of bacterial DNA into plant nuclear DNA. Virulent A. tumefaciens strains harbor a large Ti (tumor-inducing) plasmid that carries genes essential for tumorigenesis. In the present study, 13 strains (Malus pumila Mill; $A_{1-3}$, Populus monilifera; $W_{1-6}$, Populus tomentiglandlosa; $P_{1-3}$ and Rosa species; $R_1$) of Agrobacterium isolated in korean crown gall tumors and plasmids were observed in 6 strains ($W_2$, $W_3$, $W_6$, $P_1$, $P_3$ and $A_2$). The test for crown gall tumor formation was resulted only in ATCC15955 and $KW_2$ strains inoculated into the stem of sun flower and the development was observed for 4 and 6 weeks after inoculation. Above two Ti plasmids (pTi) were purified by cesium chloride-ethidium bromide density gradient centrifugation and digested with restriction enzyme and fragments of pTiATCC 15955 and $pTiKW_2$ observed by EcoR I ; 25&27, Hind III; 23&21, BamH I ; each 20 and Hpa I ; 12&27, and sizes of pTiATCC15955 and and $pTiKW_2$ calculated as 200 and 87 kbases. Octopine was isolated from tumor tissue ($W_{1-6}$ and $P_{1-3}$) and these strains confirmed as octopine type.

• KSBB Journal
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• v.7 no.3
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• pp.191-200
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• 1992

Several strains of Agrobacterium tumefaciens were isolated from soil in the Taegu area and characterized to develop some useful vector systems for higher plant genetic engineering. The selected colonies had a unique form, and strains from the colonies were capable of tumor formation on the sunflower leaf surface. They had a large plasmid. The restriction analysis showed that they were another kinds of Ti plasmic compared with C58 and Ach5. The isolated strains were identified as the nopaline type and also as biovar 1 A. tumefaciens, according to their tumor morphology, blophyslcal and biochemical characteristics. One of the isolated strains, AK204 was transformed with binary vector (pGA642), having selectable marker (Kmr, Tcr). Furthermore, maize tissue cells were transformed by cocultivation with AK204/pGA642, and the transformants were selected on the selective medium and identified using PAGE patterns of their soluble proteins.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.2
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• pp.106-109
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• 2000

Vascular endothelial cells (EGs) are usually difficult to culture to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement(ECGS). The expression of VEGF by HUVEC tansfected with Vegf GENE was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS. However, when the culture medium was supplied with 2.5 ng/ml of basic fibroblast growth factor (bFGF), a synergistic effect effect of VEGE and bFGF was observed. In this case, the final cell density was recovered was recovered up to about 78% of maxium value.

• Korean Journal of Microbiology
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• v.27 no.4
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• pp.323-333
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• 1989

Plasmid DNA molecules containing strong promoter upstream from IS50L or IS50R, the two insertion sequences that flank Tn5, were constructed to amplify the synthesis of Tn5-encoded polypeptides. When proteins made by cells that contain these plasmids were analyzed on polyacrylamide gels, enhanced synthesis of IS50R polypeptides could be detected. Synthesis of this polypeptide apparently is initiated within the large open reading frame of this element. In addition, the stability of IS50L-and IS50R-encoded polypeptides was analyzed. It was found that IS50L polypeptides are relatively unstable in vivo. This instability could account for the observed inability of this element to promote transposition.

• Genomics & Informatics
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• v.4 no.2
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• pp.80-86
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• 2006

It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.

• Korean journal of applied entomology
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• v.32 no.3
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• pp.300-306
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• 1993

The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.