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DH10B菌株高效电转化条件探究 总被引:6,自引:0,他引:6
以pUC19、pECBAC1、pCLD04541DNA以及3个不同大小的BACDNA为材料,研究了E.coli DH10B菌株在5个不同脉冲电场下的转化效率。研究发现,随着DNA片段大小的增加,最高转化效率和最适场强迅速减小。利用DH10B细胞转化pUC19 DNA的最适场强是21kV/cm,而190kb BAC DNA仅为13kV/cm;在最适场强下,40kb BAC DNA的转化效率约是190kb BAC DNA的50倍。通过大量数据绘制了不同因素影响下转化效率的变化曲线,优化了E.coli DH10B菌株电转化条件,为质粒的重组转化以及大片段基因组文库的构建奠定了基础。 相似文献
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电转化法提高平连载体DNA转化效率的研究 总被引:3,自引:0,他引:3
探讨了电转化过程中一些影响平连载体DNA转化效率的因素,并与化学法进行了比较,由此建立了优化的电转化条件。结果显示,在OD600值0.72-0.78收获细胞时,可使平连载体DNA的转化效率达到5×106转化子/μgcD-NA,较化学法高102-103倍。同时,降低连接产物的盐浓度,对于电转化成功及提高转化率也至关重要 相似文献
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介绍目前常用的双歧杆菌感受态细胞制备方法,并系统研究影响双歧杆菌电转化效率的关键因素。通过研究,菌体在4oo为0.3-0. 5时收集以制备感受态细胞,制备好的感受态细胞应尽早用于电转化;最佳电场强度为12.5 kV/cm;转化后的细胞复苏培养2 h为佳。感受态细胞的转化效率可达103 CFU/μg DNA。 相似文献
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【背景】嗜热链球菌AR333是本实验室从发酵乳中筛选出的一株高产活性胞外多糖乳酸菌。【目的】建立嗜热链球菌AR333高效电转化体系。【方法】通过单因素试验和Box-Behnken响应面法优化电转化条件。【结果】嗜热链球菌AR333最优电转化条件为甘氨酸浓度8.3g/L,OD_(600)为0.8,10%甘油(体积比)和0.5 mol/L蔗糖的电转缓冲液,pIB184质粒80 ng,电场强度14 kV/cm,0.4 mol/L山梨醇、2 mmol/L CaCl_2和20 mmol/L MgCl_2的LM17复苏培养基,复苏时间5 h。【结论】在最优电转化条件下,嗜热链球菌AR333电转化效率达到3.68×10~5 CFU/μg-DNA,比优化前提高了14倍,实现了嗜热链球菌AR333的高效遗传转化,为其功能解析和基因工程改造奠定基础。 相似文献
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外源载体高效转化肺炎克雷伯菌的新途径 总被引:1,自引:0,他引:1
研究介绍了提高Klebsiella pneumoniae电转化效率的新途径,即直接从固体平板上收集K.pneumoniae菌落制备电转化感受态细胞,完全不同于传统的试验方法。试验菌株为野生型K.pneumoniae NTUH-K2044和magA—突变型菌株。将大小不同的质粒pIP843T、pIP843TdhaB、pIP843TdhaT电转化K.pneumoniae,计算电转化效率。电转化试验结果表明:K.pneumoniaeNTUH-K2044固体菌电转化效率高达2×105±300转化子/μgDNA,而其液体菌电转化效率仅为150±10转化子/?gDNA;其magA—突变株固体菌的转化效率最高,可以达到3.4×107±500转化子/μgDNA,比液体菌电转化效率提高了104倍。同时发现质粒大小对电转化效率并没有明显影响。此外,激光共聚焦显微镜观察发现固体平板和液体培养基中的菌体存在形态学方面差异,推测固体培养菌电转化效率的显著提高和形态学方面的表现可能具有一定的相关性。 相似文献
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棒状类细菌电击转化中多种条件对转化效率的影响 总被引:9,自引:0,他引:9
以质粒PXZl0145电击转化不同棒状类细菌菌株,研究了影响电击转化效率的诸个因素,在含4%甘氨酸的培养基中生长至对数前期的菌体最适用于电击转化,当以同源DNA进行电击转化时,1.μgDNA中转化效率最高可达到8×1O6转化子,但用异源DNA时,转化效率要比前者低102~103倍。 相似文献
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Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation 总被引:7,自引:0,他引:7
AIMS: To optimize the conditions for electroporating foreign plasmid DNA into Lactobacillus acidophilus ATCC 43121. METHODS AND RESULTS: The conditions of electroporation were optimized to improve the transformation efficiency. Plasmid pNZ123 containing multicloning site and chloramphenicol resistance was employed to construct a cloning vector. The optimum electroporation conditions for the maximum transformation efficiency were a pulse strength of 12.5 kV cm(-1), a pulse number of 10, a pulse interval of 500 ms, and pNZ123 plasmid DNA concentration of 25 ng microl(-1). Under the optimum conditions the transformation efficiency of L. acidophilus ATCC 43121 was 1.84 +/- 0.13 x 10(4) (+/- standard error of measurements) CFU per mug of plasmid DNA. Other strains of L. acidophilus showed transformation efficiencies ranging from 1.38 +/- 0.02 x 10(4) to 9.32 +/- 0.54 x 10(4) under these conditions. A green fluorescent protein (GFP) was successfully expressed and detected by fluorescence microscopy when the pKU::slpA-GFP, pNZ123 containing GFP gene, was transformed in L. acidophilus ATCC 43121 under the optimum conditions. CONCLUSIONS: The results suggest that electrical parameters, antibiotic concentration, and host specificity play important roles to determine transformation efficiency of lactobacilli. The optimum conditions for the transformation of L. acidophilus ATCC 43121 may be applied to improve transformation efficiency of other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized conditions for electrotransformation may provide a mean to improve the introduction of foreign DNA into L. acidophilus to be used as a vehicle for a heterologous protein expression. 相似文献
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Aims: The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 102 –105 transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results: MgCl2 and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg2+ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg2+ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg2+ . To maintain the stability of the cell membranes, Mg2+ was replaced with high concentrations of sucrose, from 272 to 408 mmol l−1 . With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 106 transformants per microgram DNA.
Conclusions: Mg2+ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg2+ is absent.
Significance and Impact of the Study: A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg2+ can be used for other nuclease-producing organisms. 相似文献
Methods and Results: MgCl
Conclusions: Mg
Significance and Impact of the Study: A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg
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Takako Yoshida Yuko Ayabe Masae Horinouchi Hiroshi Habe Hideaki Nojiri Toshio Omori 《Biotechnology letters》2001,23(10):787-791
The transformation efficiency of Methylobacillus sp. strain 12S, using electroporation, was unaffected by the growth phase of the cells but competent cells grown at 21 °C had a 1.9 × 103 times higher transformation efficiency than those grown at 30 °C. Heat shock treatment further increased the transformation efficiency up to 7 times. 相似文献
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We have found that a significant difference exists in transformationefficiency between the crp+/crp isogenic pair of strainsof Escherichia coli, with the efficiency being much higher incrp than in crp+. The ratio of transformation efficiencybetween crp+ and crp strains depends very little on theplasmid size. This observation suggests that the differenceof the transformation efficiency is due to mechanisms otherthan a crp-regulated endonuclease. The crp gene is one of thefirst specific genes that have been shown to affect transformationefficiency. 相似文献
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杜氏盐藻电击转化方法的系统优化 总被引:1,自引:0,他引:1
本研究系统分析了盐藻生长状态、电击条件、电击缓冲液成分和质粒浓度等条件对电击转化效率的影响。实验结果表明:正常接种后培养7d对数生长中期的盐藻细胞,在25μF、0.8kV的电击条件下加入终浓度为10μg/mL的质粒可使盐藻电击转化效率达到1.85‰;电击缓冲液中加入0.4mol/L的甘油可使转化效率显著提高至2.03‰(P<0.05)。在上述优化电击体系下,运用3种不同质粒分别转化盐藻细胞后获得的转化效率无显著差异。通过对电击转化中相关因素的优化,本研究建立了一种适用于杜氏盐藻的高效稳定的电击转化体系,为杜氏盐藻的转基因研究提供有效方法。 相似文献
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Ki Jun Jeong Hyun Sook Lee Sang Yup Lee Yong Keun Chang 《Biotechnology and Bioprocess Engineering》1998,3(1):48-49
A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a
high transformation efficiency. The highest efficiency of 1.6 × 106 transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD600 of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF. 相似文献
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Two chimaeric genes, containing the promoter of the 35S gene of cauliflower mosaic virus coupled to neomycin phosphotransferase (35S-NPT-NOS) or to hygromycin phosphotransferase (35S-HPT-NOS) have been stably transferred to maize (Zea mays cv. Black Mexican sweet) cells by electroporation. Transformation frequencies of 7.6×10-4 and 8×10-4, respectively, (based on the number of surviving cells that divided) were obtained with four pulses of 1 ms duration using 400 V capacitive discharge. Cells transformed to kanamycin-resistance and hygromycin-resistance subsequently multiplied to form callus. Southern blot analysis demonstrated the integration of the selectable marker genes, neomycin or hygromycin phosphotransferase, with single or multiple copy numbers. The blots from DNA of hygromycin-resistant calli also suggested the formation of plasmid concatemers. 相似文献
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Kinga Kania Anna Levytska Anna Drożak Borkowski Andrzej Działak Paweł Maksymilian Zienkiewicz 《Biochemistry and Biophysics Reports》2022
In this study, we have shown that transformation efficiency of Coccomyxa subellipsoidea C-169 obtained by electroporation can be significantly increased by either supra- or sub-optimal growth temperatures. 相似文献
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Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 105 transformants μg?1 DNA for WB800, and 2·10 × 105 transformants μg?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains. 相似文献