• 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.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• KSBB Journal
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• v.5 no.3
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• pp.247-253
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• 1990

These studies were carried out to obtain the transformant from carrot cells by using binary vector pGA472 with NPT II gene to confer kanamycin resistance in the plant cells. The binary vector pGA472 was mobilized from E. coli MC1000 into A. tumefaciens strains isolated in the Korea, C23-1. K29-1, and disarmed Ti-plasmid PC2760, and A28l using a tri-parental mating method with E. coli HB101/pRK2013. Transconjugants, C23-1/pGA472, K29-1/pGA472, PC2 760/pGA472 and A28l/pGA472 were obtaind on the minimum AB media containing tetracycline and kanamycin, were comfirmed to hold the Ti-plasmid and pGA472 binary vector on the 0.7% agarose gel. Transformed carrot calli were initiated on the MS media supplemented with l00$\mu\textrm{g}$/ml kanamycin and 250$\mu\textrm{g}$/ml carbenicillin after co-cultivation of carrot explant and transconjugant Agrobacteria. Selected callus was grown vigouousley for subculture on the medium containing 100$\mu\textrm{g}$/ml kanamycin, thus indication that the selected callus was transformed with NPT II gene.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.433-438
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• 1991

The plasmid pSY130-14 for the high production of phenylalanine is a temperaturecontrollable expression vector composed of the $P_R$ and the $P_L$ promoter and a temperature sensitive repressor, $cI_{857}$ of bacteriophage lambda. Strain AT2471 harbouring plasmid pSY13O- 14 is induced the phenylalanine production by shifting up the incubation temperaure to $38.5^{\circ}C$. Plasmid stability of E. coli AT2471 harbouring pSY130-14 was very low, it was about 30% after 48 h cultivation at $38.5^{\circ}C$ without kanamycin. The plasmid disappeared immediately at $40^{\circ}C$ without kanamycin, and at $40^{\circ}C$ adding kanamycin, the plasmid stability decreased at the beginning, but rose with the extension of the culture time. For the improvement of plasmid stability, the plasmid obtaind was designated as pSY15O-1 by changing origin region (ori) pACYC 177 of pSY130-14 for ori pSC101. E. coli AT2471 harbouring pSY150-1 was stable at $38.5^{\circ}C$ without tetracycline, and the plasrnid stability was about 40% after 48 h cultivation at $40^{\circ}C$.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.271-279
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• 1994

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase.

• Korean Journal of Microbiology
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• v.22 no.4
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• pp.249-255
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• 1984

To develop the host-vector system for industrial Coryneform bacteria that seemed to be the most suitable microorganisms for molecular breeding of genes involved in the production of amion acids, nucleotides, and other products of industrial interest, broad host range E. coli plasmid R 1162 DNA was transformed into Brevibacterium ammoniagenes and the plasmids pKU6 isolated from a transformant was physically characterized. All other plasmids from the transformed cells except pKU6 exsisted as multimeric forms in Brevibacterium ammoniagenes. The plasmid DNA was retransformed into Corynebacterium glutamicum with a high frequency ($1.32{\times}10^{-1}$ per cell) and maintained stably both in Brevibacterium ammoniagenes and Corynebacterium glutamicum after 100 generations of cultures with 25-30 copy number per cell. The size of both plasmid pKU6 and plasmid R1162 were the same and restriction maps by EcoR I, Ava I, Pst I, Pvu II and Hinc II were also similar.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.125-131
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• 1986

In order to obtain the optimum conditions for intact yeast cell transformation in the various yeast host-vector systems, 3 yeast plasmid vectors, YRp7, YEpl3 and YIp5 were introduced into 5 yeast hosts, Saccaromyces cervisiae Dl3-1A, DKD-5D, DBY-746, MC-16 and S2022D with various transformation conditions, and plasmid stabilities in all the transformants were also observed. The highest transformation frequencies in all the host-vector system were obtained in the 16 hour Cultured cell (5.4 $\times$ 10$^6$ - 2.4 $\times$ 10$^8$cells/$m{\ell}$) treated with 0.1-0.2 M lithium chloride in 0.1 M tris-HCl (pH 7.6), 35% polyethylene glycol 4000, and heat-shocked at 42$^{\circ}C$ for 5 minutes after 60 minutes of induction. The intact cell transformation got more transformation frequency in DKD-5D (YRp7) and DBY-746 (YEpl3) than protoplast transformation, but reverse tendency was observed in DKD-5D (YEp13) and Dl3-lA (YRp7). The transformants, D13-1A (YRp7) and DKD-5D (YRp7) were very unstable in selective medium, with 80 to 85% of the transformants losing the plasmid after 70 generations, but the transformants, DKD-5D (YEpl3) and DBY-746 (YEpl3) were quite stable, with 35% of the transformants losing the plasmid.

• Journal of Pharmaceutical Investigation
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• v.26 no.4
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• pp.291-297
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• 1996

Liposomes of $pSV-{\beta}-Galactosidase$ vector plasmid DNA with various lipid composition were prepared by the thin-film method. Size distribution, shape and the efficiency of plasmid DNA encapsulation were investigated. Effect of sonication time on the plasmid DNA entrapment in liposomes and stability at $4^{\circ}C$ were also examined. Sizes of neutral liposomes were about 100-200 nm and above $1\;{mu}m$, and those of cationic liposomes were about 400-600 nm and above $1\;{mu}m$. Shapes of liposomes entrapped plasmid DNA were spherical. Proper sonication time for better entrapment was below 15 minutes and stability at $4^{\circ}C$ was decreased rapidly after 1 day. Plasmid DNA entrapments of complex liposomes of various lipids were higher than those of liposomes made from one sort of lipid. Plasmid DNA entrapments of cationic liposomes were higher than those of neutral liposomes.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.263-267
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• 1993

Transgenic animals have become an important tool in the basic and applied sectors of genetic and biomedical sciences. In particular transgenes provide clear-cut markers in the spatial and temporal analysis of developing embryos for the understanding of developmental mechanisms. For the long-term use of plasmid vector in a particular purpose it would be necessary to develop one's own vector system which can be properly expressed in eukaryotic system. Plasmids were constructed from ori region of pUC19 and early region of SV40 through various steps. LacZ gene coding for $\beta$-galactosides was fused to early gene of SV40 in translational in-frame. Poly(A) tailing site of SV40 was inserted at the 3' lacZ so that initiation, elongation and terminatin be controlled by SV40 transcription (pSS4). Biological function of the constructed pSS4 was demonstrated via microinjection of the plasmid into fertilized loach eggs and subsequent detection of $\beta$-galactosidase in developing embryos. The result indicate that the newly constructed pSS4 is functional in a eukaryotic system in vivo. Thus pSS4 may be used as an efficient tool for the study of embryogenesis and a basic carrier for various genes for animal transgenesis.

• 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.