• 한국식품영양학회지
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• 제5권2호
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• pp.77-83
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• 1992

The optimum conditions and mechanisms for the plasmid-mediated genetic transformation of intact cells of Bacillus brevis Pl76-2, an extracellular protein producing bacterium by electroporation were investigated. It was found that pUB110 Plasmid DNA can be introduced into intact bacterial cells by electroporation. The frequency of transformation by this electroporation system depended upon the initial electric field strength, the capacity of the electric discharge capacitor, growth stage, number of successive pulses and composition of electroporation buffer. It was effective for transformation that cells were harvested, washed and resuspended with HSM [7M HEPES(PH 7.4), 272mM sucrose, 1 mM MgCl2] electroporation buffer when cell growth was attained to 1.2 at OD660. A maximum frequency of transformation of 2.40$\times$104 transformants per$\mu$g plasmid DNA was obtained by two succesive Pulses with an initial electric field strength of 12.5kV/cm and with a capacitance of 7.3uF.

• 미생물학회지
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• 제36권2호
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• pp.109-111
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• 2000

Lactobacillus casei ssp. casei NCIB 4114 균주로부터 3,5kb의 플라스미드를 분리하여, 이 플라스미드를 함유하는 Escherichia coli-Lactobacillus 셔틀 백터(shuttle vector)들을 만들었다. tu틀 벡터들은 모두 electroporation에 의해 성공적으로 형질전환 되었다. Electroporation의 최적조건은 벡터DNA 1$\mu$g당 $2{\times}10^5$ 형질전환체의 효율이었고, 이들 벡터의 성공적 도입은 이들 벡터의 유산균에서의 음식등급 벡터로의 사용 가능성을 제시하였다.

• Applied Biological Chemistry
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• 제38권5호
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• pp.371-375
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• 1995

식물 병원성 사상균 F. oxysporum과 R. solani에 대하여 우수한 길항력을 갖는 Pseudomonas (P.) fluorescens를 작물 근권으로 부터 분리하여 생리, 생화학적 특성을 조사하였다. 이들 분리 근권길항 미생물중 한 균주인 Ps70과 plasmid pSV2-neo를 이용하여 electroporation에 의한 길항 미생물 P. fluorescens의 transformation 가능성과 최적 조건을 조사하였다. 그 결과 10% glycerol을 P. fluorescens buffer로 사용하여 2.5kV의 voltage, $200{\Omega}$의 resistance에서 최적의 electrotransformation 효과를 보였다. 또한, 이균주에 크기가 서로 다른 plasmid를 electroporation하여 transformation 효과를 비교한 결과 voltage, electroporation buffer의 조성, 그리고 resistance (time constant)가 transformation의 효과를 증진하는데 주요한 역할을 하는것으로 나타났으며, 또 다른 P. fluorescens 균주에 같은 실험을 반복한 결과 유사한 electrotransformation 효과를 보였다.

• Journal of Plant Biotechnology
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• 제3권3호
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• pp.123-129
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• 2001

Electroporation of microcalli and embryonic axes of a Brazilian Indica rice cultivar was performed. Some parameters influencing the recovery of transformed callus have been defined through transient npt II expression. Such parameters included the presence of light during incubation of microcalli used as target for electroporation, heat shock at 45$^{\circ}C$, macerozyme pre-digestion of target tissues and the number of pulses during electroporation. Transgenic calli were obtained from embryonic axes after electroporation with plasmid pDM302, which encodes the gene phosphinotricin acetyl transferase (bar) under the control of Act-1 promoter. Integration of the introduced gene into the genome was demonstrated by Southern blot hybridization.

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권1호
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• pp.48-49
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• 1998

A protocol for the transformation of Klebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain high transformation efficiency. The highest efficiency of 1.6$\times$106 transformants per $\mu\textrm{g}$ DNA(pBR322) could be obtained by electroporation of K. oxytoca cells prepared at the OD600 of 0.2 with 1.25$\mu\textrm{g}$ DNA at the filed strength of 2.5kV, the parallel resistance of 200$\Omega$ and capacitance of 25$\mu$F.

• Journal of Microbiology
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• 제35권3호
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• pp.181-187
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• 1997

Filamentous cyanobacteria are an ecologically important group of bacteria because they are able to provide both organic carbon fixed nitrogen that can support the nutritional requirements for other microorganisms. Because of their prokaryotic nature, they can also be used as potentially powerful model systems for the analysis of oxygenic photosynthesis and nitrogen fixation. Gene transfer is an indispensable procedure for genetic analysis of filamentous cyanobacteria. Electroporation was used to introduce foreign DNA into cyanobacterial cells. In experiments designed to optimize the electroporation technique, the effects of the field strength (amplitude of pulse) and time constant (duration of pulse), DNA concentration and host restriction/modification of DNA on the efficiency of electro-transformation were investigated. The results of this research revelaed that a high voltage pulse of short duration was effective for the electro-transformation of Anabaene sp. M131. The maximal number of transformants was obtained at 6 kV/cm with a pulse duration of 5 msec. The efficiency of electro-transformation was also sensitive to concenetration of DNA; even small amounts of DNA (0.01 .mu.g/ml) were able to gie a large number of transformants (1.0 * 10$\^$3/ cfu/ml).

• Mycobiology
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• 제38권4호
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• pp.331-335
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• 2010

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/${\mu}g$ of DNA in $1{\times}10^7$ protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

• KSBB Journal
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• 제6권4호
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• pp.385-388
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• 1991

자란(B. striata)의 미성숙종자로 부터 유도한 embryogenic callus를 현탁배양하고 이와같은 embryogenic cell suspensions로부터 분리한 원형질체에 electroporation 방법을 사용하여 reporter genes이 들어있는 pBI121 plasmid DNA 를 도입하고 그 발현을 확인하였다. 배양된 세포에 있어서 GUS의 활성은 plasmid DNA양의 증가와 함께 높게 나타났으며, 200-300 voltage/1180 uF에서 GUS의 활성 및 원형질체의 생존율(viability)이 가장 좋았다.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.222-228
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• 2001

Vibrio metschnikovii strain RH530 is a non-pathogenic, industrially-important alkaline protease producer which has been isolated from wastewater. In this paper, we report on the transformation of this strain by using the method of electroporation. A field strength of $7.5\;kVcm^{-1}$ and $25\;{\mu}F$, and using a 0.2-cm cuvette, appeared to be the optimal conditions for electroporation of the cells with the recombinant pSBCm plasmid carrying the vapK alkaline protease gene and the ColE1 replicon. Cells were subjected to osmotic shock in order to remove extracelluar DNase, and adding 200 mM of sucrose to electroporation buffer cells showed an increased transformation efficiency. Maximum efficiency of transformation was obtained at an early exponential growth phase. Using all of the conditions mentioned above, we routinely obtained a transformation efficiency of more than $10^4{({\mu}g\;plasmid\;DNA)}^{-1}$. The stability of the plasmid pSBCm in V. metschnikovii RH530 was 25% after 18h of growth (27 generations) in the medium without antibiotic selection. The insertion of the par locus to the pSBCm increased the stability of the plasmid up to 42% without selective pressure. The increase in plasmid stability was accompanied by the increase in the productivity of alkaline protease in the recombinant V. metschnikovii strain RH530. Determining optimal conditions for the transformation of the industrially-important, nonpathogenic Vibrio strain, and the improvement of plasmid stability by introducing the par locus into the high copy number plasmid vector, will allow the development of procedures involved in the genetic manipulation of this strain, particularly for its use in the production of industrial enzymes such as alkaline protease.

• 약학회지
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• 제40권1호
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• pp.59-64
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• 1996

Gram-positive bacteria, Bacillus subtilis BR151 and Staphylococcus aureus RN4220 were transformed with high efficiency by electroporation. The cells were incubated until late log phase, washed three times with 10% glycerol, 1mM HEPES, 12% sucrose and resuspended to $10^{10}{\sim}10^{11}cfu/ml$, then stored at -$70^{\circ}C$. Transformation efficiency of B. subtilis BR151 was $1.03{\times}10^7cfu/{\mu}g$ with cells washed with 10% glycerol and electroporated by 15KV/cm, 0.7msec pulse with pUB110. Transformation efficiency of S. aureus RN4220 was $4{\times}10^6cfu/{\mu}g$ with cells washed with 1mM HEPES + 10% glycerol and electroporated by 15KV/cm, 2.5msec pulse. The number of total transformants was 1000 when B. subtilis BRI51 was transformed with 100ng pUB110 DNA and the number of total transformants was 9000 when S. aureus RN4220 was transformed with 10ng pUB110 DNA