• 한국미생물·생명공학회지
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• 제29권1호
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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.

• 한국미생물·생명공학회지
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• 제14권2호
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• pp.155-160
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• 1986

E. coli-Yeast shuttle vector YEp 13에 B. amyloliquefaciens의 $\alpha$-amylase gene을 cloning하여 얻은 hybrid plasnidlasmid를 E. coli를 숙주세포로 하여 형질을 발현시켰다. 형질전환주의 $\alpha$-amylase 활성 측정 결과, B. amyloliquefaciens에 대해 20-30%의 상대 활성도를 나타내었다. 형질전환주의 경우 생성된 $\alpha$-amylase의 60-65%가 periplasm에 축적되었으며 세포 외부로의 분비는 없었다. Hybrid DNA를 agarose-gel 전기영동으로 조사한 결과 그 크기가 다른 다수의 hybrid DNA가 확인되었으며 pHA28의 plasmid (a)(Fig.3)에서만 $\alpha$-amylase 활성을 나타내는 것으로 밝혀졌다 pHA28의 plasmid(a)의 크기가 YEp 13 plasmid DNA보다 작은 것은 YEp13 plasmid에서 yeast gene부분이 deletion된 결과로 추측되었다.

• KSBB Journal
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• 제5권3호
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• pp.247-253
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• 1990

고등식물의 형질전환용 유전자운반체로써 가장 많이 사용되고 있는 Ti-plasmid를 이용해서 당근세포를 형질 전환시키기 위한 연구의 일환으로 Agrobacterium spp를 helper로 이용하여 NPT II gene를 함유하고 있는 binary vector GA472를 당근세포에 삽입시켜 kanamycin에 대해 저항성을 나타내는 세포주를 선발하고자 본 연구를 수행하였다. 국내 토양에서 선발한 A.tumefaciens 2종과 disarmes된 PC2760 그리고 hypervirulent균주인 A281에 tri-parental mating 방법에 의해서 binary vector인 pGA472을 도입하여 transconjungants인 A. tumefaciens c-23-1/pGA472,K29-1/pGA472, PC2760/PGA472 그리고 A281/pGA472를 획득하였다. Transconjungants는 plasmid의 분리, 정제방법에 의해서 추출한 후 0.7% agarose gel 상에서 관찰해 본 결과 4균주 공히 NTPII gene이 삽입된 pGA472와 Ti-plasmid를 함유하고 있는 것을 확인 하였다. 확인된 conjugant와 당근정상조직을 동시배양방법에 의해서 형질전환을 유도한 후 정상조직은 전혀 생존이 되지않은 kanamycin에 대해서 저항을 나타내는 callus를 선발할 수 있었다.

• 한국미생물·생명공학회지
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• 제19권5호
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• pp.433-438
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• 1991

Phenylalanine 고생산용 plasmid pSY130-14는 $\lambda$-phage 유래 온도감수성을 가지고 있으므로 $cI_{857}$ repressor와 PL과 PR을 온도를 올려 phenylalanine의 생산을 유도한다. pSY130-14를 갖는 E.coli AT2471은 kanamycin 무첨가, $38.5^{\circ}C$, 48시간에서 약 30%로 plasmid가 없어지며 첨가에서 안정성이 떨어졌다가 배양시간과 더불어 올라갔는데, 이것은 생육에 피요한 kanamycin gene과 ori만이 남는 것으로 생각된다.

• 미생물학회지
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• 제32권4호
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• pp.271-279
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• 1994

${\alpha}$-Amylase와 glucoamylase를 동시에 안정하게 분비하여 전분을 일단계로 직접 에탄올로 발효시킬수 있는 효모 균주를 개발하기 위하여 glucoamylase를 분비하는 Saccharomyces diastaticus hybrid 균주에 쥐의 침샘 유래의 ${\alpha}$-amylase cDNA 유전자를 plasmid vector를 이용하여 도입하였다. 이 균주로부터 효소생산에 필요한 유전자를 잃어버림이 없이 안정하게 분비할 수 있도록 하기 위하여 $\alpha$-amylase 유전자를 효모의 염색체에 삽입시키기 위한 integrating plasmid vector인 YIpMS$\Delta$R(LEU2)를 제작하였다. 이 vector의 효모형질전환에 있어 원형(circular)상태와 제한 효소 XbaI으로 처리된 직선화된(linearized) 상태의 두가지 형태를 비교한 결과 형질전환 효율에서나 형질전환체내의 $\alpha$-amylase 유전자 보유정도가 모두 직선화된 형태의 경우가 원형상태의 경우보다 높았다. Linearized vecotr를 가진 효모 형질전환체에서의 유전자 발현 안정도는 세포분열을 거듭할수록 episomal vecotr에 의한 효모 형질전환체에서의 발현 안정도보다 우수하게 나타났다. 또한 이 linearized vector를 가진 형질전환체는 $\alpha$-amylase와 glucoamylase를 동시에 분비하여 glucoamylase만 분비하는 원균주보다 2배 이상의 전분분해력을 보였다.

• 미생물학회지
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• 제22권4호
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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.

• 한국미생물·생명공학회지
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• 제14권2호
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• pp.125-131
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• 1986

각 효모 숙주 및 vector에 따라 lithium염 처리에 의한 생효모형질전환 최적 조건을 얻기 위하여 5종의 효모 숙주(S. cerevisiae Dl3-lA, DKD-5D, DBY-746, MC-16 및 S2022D)에 3종의 효모plasmid vector(YRp 7, YEp 13 및 YIp 5 )의 형질전환 실험과 아울러 이들 각 형질전환체내에서 도입된 plasmid들의 안정성을 조사하였다. lithium 염의 경우 LiCl가 Li-acetate에 비하여 좋은 효과를 보였으며 LiCl처리에 의한 최적 형질전환 조건은 각 숙주-vector계에 있어서 공히 균체 배양 시간은 16시간 (5.4 $\times$ $10^6$ - 2.4$\times$$10^8$cells/$m{\ell}$ ) 내외, LiCl의 농도는 0.1-0.2M, PEG(4000)의 농도는 35%, induction시간은 60분 내외 열처리는 42$^{\circ}C$에서 5분간, LiCi처리 buffer는 0.1M tris-HCl(pH 7.6)에서 가장 높은 형질전환 빈도를 보였다. 한편 Protoplast 형질전환법과 형질전환빈도를 비교해 본 결과 DKD-5 D(YEp13)과 Dl3-lA(YRp7)의 경우는 protoplast법이 DKD-5D(YRp 7), 및 DBY-746(YEp 13)에서는 LiCl처리법이 형질전환 빈도가 높았으며 MC-16(YEp 13)의 경우는 양방법에서 공히 비교적 낮은 빈도를 보였다. 각 형질전환체내에서의 도입된 plasmid의 안정성은 선택배지에서 배양했을 경우- YRp 7이 Dl3-lA 및 DKD- 5D 숙주에서 70세대후에는 80-85%가 유실되었으나 YEp13은 DKD-5D 및 DBY-746에서 35%밖에 유실되지 않았으며 MC-16숙주에서는 55% 유실로서 비교적 안정하였다. 또한 비선택배지에 배양시에는 선택배지에 배양했을 경우 보다 안정성이 다소 낮았으나 같은 경향은 보였다.

• Journal of Pharmaceutical Investigation
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• 제26권4호
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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.

• 한국가축번식학회지
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• 제17권3호
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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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• 제18권1호
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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.