• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.687-690
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• 1999

Bacillus stearothermophilus No. 236 isolated from the soil is a strong xylan degrader producing all the xylanolytic enzymes. However, the strain was discovered to be highly intractable to its transformation. In the present study, we have developed a reliable method for transformation of B. stearothermophilus No. 236 by a systematic examination of several factors which might have an influence on the efficiency of electrotransformation. Notably, we found that the most critical factor influencing the transformation efficiency (TE) was the incubation temperature after pulsing, with its optimum incubation of $37^{\circ}C.\; At\; 50^{\circ}C$, the optimum growth temperature of the B. stearothermophilus strain, the transformants could not be obtained at a recognizable level. The combination of field strength of 7.5 kV/cm along with pulse duration of 10 msec (resistance of $400{\Omega}\; and\; capacitance\; of\; 25{\mu}F$) was shown to be the best electrical parameters at the incubation temperature of $37^{\circ}$. A higher TE was obtained when the cells were harvested at an early-exponential phase. Twenty percent of PEG-8000 in a suspension buffer and an addition of 0.1% glycine in the growth medium resulted in about 4-fold and 3-fold increases in TE, respectively. We also found that the plasmid DNA which had been cycled through the host B. stearothermophilus cells enhanced TE by one order of magnitude higher. Under the presently described conditions, $2.5{\times}10^{5} transformants per ${\mu}g$ DNA was attained.

• 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).

• Plant Biotechnology Reports
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• 제5권2호
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• pp.147-156
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• 2011

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

• Mycobiology
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• 제47권1호
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• pp.59-65
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• 2019

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (${\leq}0.1%$) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per $10^6$ YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

• 한국수학교육학회지시리즈B:순수및응용수학
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• 제30권4호
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• pp.347-355
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• 2023

For numerical evaluation of Cauchy principal value integrals, we present a simple rational function with a parameter satisfying some reasonable conditions. The proposed rational function is employed in coordinate transformation for accelerating the accuracy of the Gauss quadrature rule. The efficiency of the proposed rational transformation method is demonstrated by the numerical result of a selected test example.

• 한국작물학회지
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• 제58권2호
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• pp.119-127
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• 2013

The interaction between Agrobacterium and soybean has been studied at the transcriptome level but not at the metabolic level. However, it is necessary to investigate the difference in metabolites between susceptible and non-susceptible cultivars for high efficiency transformation. We investigated the difference in metabolites from sonicated soybean cotyledons of Korean cultivars and Bert cultivar. To identify difference in metabolites, sonicated extracts were analysed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The soybean cultivars were classified by susceptibility using green fluorescent protein expression. We found a difference in metabolites between the high susceptible and low susceptible cultivars. The FT-ICR/MS experimental m/z data of different metabolites were compared with theoretical m/z in KNApSAcK database. The candidate list was made using KNApSAcK and focused on phenolic compounds. These candidate metabolites are speculated to influence factors in the interaction. This list of candidates may be useful to investigate the interaction between Agrobacterium and plants to increase transformation efficiency.

• 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

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.156-163
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• 1995

Bacillus sphaericus 1593 is pathogenic to the larvae of a number of mosquito species that are known as important vectors for the transmission of certain human and animal diseases. As a preliminary experiment for developing a multfunctional B. sphaericus 1593 as a potent antagonist, we investigated the conditions for the protoplast transformation system of B. sphaericus 1593 using the plasmid pGB215-110$\Delta$B. The protoplast of B. sphaericus 1593 were obtained most efficiency by treating the cells with 500 $\mu$g/ml of lysozyme in the SMM buffer containing 0.5 M sucrose at pH 8.0 and 40$\circ$C for 60 minutes. The cell wall was regenerated on the plate containing 1.2% agar and 0.8 M mannitol. Under the best condition for protoplast formation and regeneration established in the work the highest frequency of transformation was achieved with the 40% PEG (M.W 4,000) treatment for 15 minutes of incubation at 4$\circ$C, and subsequently for 120 minutes incubation at 30$\circ$C for phenotypic expression. The highest transformation efficiency were observed at 1.0 $\mu$g/ml of the final concentration of the plasmid DNA and the plasmids were found to be fairly stable since about 70% of the plasmids were maintained after 8 successive daily transfers onto the fresh medium.

• 식물조직배양학회지
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• 제27권2호
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• pp.95-100
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• 2000

배추에서 분리된 cytosolic glutathione reductase (BcGR1)유전자를 벼에 효과적으로 도입하기 위하여 몇 가지 실험을 수행하여 얻어진 결과를 요약하면 다음과 같다. 수정 후 3주된 미숙배에서 유도된 캘러스를 이용하여 형질전환 효율의 품종간 차이를 조사한 바 '대립벼'가 42.5%로 가장 높게 나타났다. 공동배양 배지 내에 acetosyringone (AS)이 첨가되지 않았을 경우 형질전환된 개체를 얻을 수 없었으나, 50$\mu$M을 첨가되었을 때 41.0%의 가장 높은 형질 전환효율을 나타내었다. PCR 증폭과 Southern blot 분석을 실시한바 BcGR1 유전자가 형질전환체의 게놈상에 정상적으로 삽입된 것이 확인되었으며, T$_1$세대 28개통 중 17개체의 hygromycin 저항성 유전형질이 3:1로 분리되었다.

• The Journal of Asian Finance, Economics and Business
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• 제9권1호
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• pp.299-310
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• 2022

Environmental protection has been infused into the development of numerous fields by the Chinese government. The government's implementation of green development has also shifted its focus to green transformation and governance of highpolluting companies. In the context of green and sustainable development, this study employs DEA data envelopment analysis to compare the operating efficiency of listed firms that implemented Green Mergers and Acquisitions (M&A) in China in 2018. The conclusions of this study are as follows: First, China's green M&A enterprises are unevenly dispersed among the country's east, middle, and western regions. Second, compared to before the implementation of Green M&A, operational efficiency has improved in most industries. Third, the difference in Green M&A across industries is generally favorable, showing that most organizations have improved their operational performance as a result of Green M&A implementation. In 2018, however, the gap in operating efficiency is more negative. Fourth, whereas the eastern and western areas' operational efficiency has improved as a result of Green M&A, the central region's has not. Based on this conclusion, this study makes recommendations for China's future sustainable development of heavily polluted firms.