• Korean Journal of Microbiology
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• v.28 no.3
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• pp.181-187
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• 1990

STA gene coding glucoamylase was introduced into haploid Saccharomyces cerevisiae SHY3 and polyploid Saccharomyces cerevisiae 54. We constructed the recombinant plasmid by substituting the promoter region of alcohol dehydrogenase isoenzyme I gene for that of STA gene to increase the expression of STA gene and found that the activity of glucoamylase was increased in transformants. The plasmid stability was improved remarkably when we got the STA gene into the plasmid which had centromere. The activity of glucoamylase and transformation frequency of it, however, was decreased because of low copy number. Industrial polyploid strain was transformed with the recombinant plasmid having the $2\mu$ origin of replication and STA gene. It produced more alcohol than host when fermented in liquefied starch media. The industrial strain, however, was not transformed with the autonomously replicating plasmid containing centromere.

• Archives of Pharmacal Research
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• v.18 no.1
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• pp.31-37
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• 1995

S. pneumoniae (pneumococcus) gene cloning and library construction in E. coli multicopy plasmid has been hampered, in part, by instability problems. In this study, stability of pneumococcus gene library in cosmid vector and pACYC184 was examined. Pneumococcus library in the cosmid vector pHC79 was extermely unstable that most of the recobinant clones were degenerated rapidly. Only 2 out 849 clones were stable and had appropriate insert size. Pneumococcus library in pACYC184 was also so unstable that the pneumococcal inserg and/or part of the vector were deleted. However, the instability problems seemed to be resolved when transcription teminator plasmid was employed for pneumococcus library construction.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.161.2-161
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• 1995

No Abstract, See Full Text

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.476-483
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• 1988

Yeat-Escherichia coli shuttle vectors were constructed by combination of various functional segments such as autonomous replicating sequence (ARS1), centromere region (CEN3), origin of replication of 2 $\mu$m plasmid (2 $\mu$m OR). Transformation efficiency, stability and copy number of constructed vectors were analyzed in yeast strains, SHY4(cir$^+$) containing 2 $\mu$m plasmid and NNY1(cir$^{\circ}$) without it. The results showed that centromere containing plasmids were very stable and existed at one copy per cell; fused replication system (2$\mu$m OR and ARS1) containing Plasmids were more stable and higher copy number than one replicon containing plasmids ; presence of endogenous 2$\mu$m plasmid influenced on stability and copy number of 2 $\mu$m based plasmids.

• Journal of Microbiology
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• v.34 no.4
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• pp.341-348
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• 1996

The regulation of xylE gene expression was examined by using vector promoter and construction of genetically engineered microorganisms (GEMs) for application in microcosm. When the xylE gene wsa subcloned into pBluscript SK(+) under the control of lac promoter (pTY1) in E. coli, and the expression was induced by IPTG, the enzyme activity of catechol 2, 3-dioxygenase was increased 4.7 times more than that of the crude extracts from transformants harboring pTY1. We suggest that the xylE gene has its own promoter at the upstream portion, because it was able to be expressed even in the absence of IPTG. A recombinant plasmid, pSW3a harboring the xylE gene under the T7 promotor, showed the activity of 14.5 units/mg protein, higher than that of parental strain, E. coli PYT1. The xylE gene in recombinant plasmid pSW3a was used as reporter gene for the application in microcosm ecosystem, since it was used for detection of xylE-positive clones by catechol spray on the agar plates. The pSW3a in E. coli was introduced into Pseudomonas patida to construct GEM strain, and examined for the exxpression and functional stability in microcosms.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4435-4439
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• 2012

Purpose: This study aimed to explore the role of the Twist gene in the epithelial-mesenchymal transition of ovarian cancer. Methods: An RNA interference plasmid expressing a small interfering RNA (siRNA)-targeting Twist (Twist siRNA vector) was designed, constructed, and transfected into the human ovarian cancer cell line A2780. Transfection efficiency was assessed under a fluorescence microscope. Changes in the expression of Twist mRNA in A2780 after transfection with the pGenesil Twist shRNA plasmid were analyzed through RT-PCR. MTT assays and adhesion experiments were applied to determine changes in proliferation and adhesion ability of A2870 after transfection with the Twist shRNA plasmid. Changes in the expression of the E-cadherin and N-cadherin proteins in A2780 after transfection with the Twist shRNA plasmid were analyzed using Western blotting. Result: The restructuring plasmid pGenesil-Twist shRNA was constructed successfully. After 48 h of culture, 80% of the cells expressed high-intensity GFP fluorescence and stability. The expression of Twist decreased significantly after the transfection of the Twist shRNA plasmid (P<0.05). Proliferation of the transfected Twist shRNA cells showed no difference with that of the A2780-nontransfection or A2780-si-control groups (P>0.05) but the adhesion ability of A2780 decreased dramatically (P<0.05). Expression of the E-cadherin protein increased, whereas that of the N-cadherin protein decreased compared with that in the A2780-nontransfection or A2780-si-control groups (P<0.05). Conclusion: Twist is essential for epithelial-mesenchymal transition, invasion, and metastasis of ovarian cancer.

• Journal of Microbiology
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• v.40 no.2
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• pp.140-145
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• 2002

The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMHl turned out to have a replication origin and two open reading frames (ORFs) of the putative γ-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMHl all constructed vectors were stable over 100 generations In a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a navel secretion vector, Using the $\alpha$-amylase promoter/signal sequence of B. subtilils the novel plasmid pJSN was constructed. When $\beta$-glucosidase was expressed using pJSN, we found $\beta$-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

• 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.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.213-218
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• 1986

Hybrid plasmid pEA24, shuttle vector YRp7 carrying amylase gene of Bacillus amyloliquefaciens, was transformed to yeast Saccharomyces cerevisiae, and the expression of B. amyloliquefaciens amylase gene in yeast was investigated. The frequency of transformation to S. cerevisiae DBY747 with YRp7 was increased by treatment of 40% polyethylene glycol (MW 4, 000), PH 7.0, at 3$0^{\circ}C$, and by regeneration used 2% top agar. The amount of cellular amylase activity produced by S. cerevisiae containing pEA24 was 2% of that secreted from B. amyloliquefaciens, but in case of S. cerevisiae transformant, the amylase secreted was not detected. A comparison of genetic stability of pEA24 and YRp7 plasmids in yeast was carried out by cultivation of transformants in tryptophan-supplement-medium. The pEA24 plasmid was more unstable than YRp7 in S. cerevisiae. The size of pEA24 extracted from S. cerevisiae transformants was found to be identical with that from E. coli transformants by agarose gel electrophoresis.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.659-664
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• 1995

To improve stability of recombinant DNA pLC1 encoding endoglucanase gene and pGL1 encoding $\beta $-glucosidase gene, DNA fragments of genes coding endoglucanase and $\beta $-glucosidase were cloned within the recA gene on a pDR1453, and the pDRE10 and pDRG20 of recombinant plasmids were integrated into the recA gene on the E. coli 1100 chromosomal DNAs. The stability of inheritance was completely maintained in E. coli 1100; Transformants E. coli 1100/pDREIO and pDRG20 were expressed well by recA promoter and increased endoglucanase and $\beta $-glucosidase activities. This method can be used as a model to improve the stability of recombinant plasmid in large scale culture.