Rapid transfer of low copy-number episomal plasmids fromSaccharomyces cerevisiae to Escherichia coli by electroporation

A simple and reproducible method for transferring low copy-number episomal plasmids from yeast toEscherichia coli has been developed. Although slightly more time-consuming than direct transfer methods, which are effective with high copy number plasmids, the method is significantly faster than methods that require purification of yeast DNA. Plasmid DNA is released from yeast cells during brief treatments involving grinding with glass beads and heating. The treated yeast are cooled, electrocompetentE. coli is added, the mixture is electroporated, and transformants are selected using standard conditions forE. coli electrotransformation. The procedure typically yields sufficient transformants for most applications.     

Production of herbicide-resistant transgenic maize plants using electroporation of seed-derived embryos

The improvement of commercial maize lines via biotechnological approaches is limited by the lack of a transformation system that is tissue culture free. In this paper, the development of a genetic transformation system is presented using electroporation for gene delivery and seed-derived embryo as the gene target. Plasmid DNA (pBARGUS), which contained the selectablebar gene for resistance to the herbicide Basta and the screenablegus gene, was delivered into enzymatically wounded mature maize embryos via electroporation. Transformed plants were identified by their ability to grow on a selective medium containing 30 mg/L of phosphinothricin. Southern hybridization, plant resistance to the application of Basta, GUS expression, and segregation analysis indicated that a functionalbar gene had integrated into the maize genome and was inherited in a mendelian fashion by the progeny.     

CAC2634-disrupted mutant of Clostridium acetobutylicum can be electrotransformed in air

Aims: To simplify the electrotransformation process of Clostridium acetobutylicum, which currently needs to be performed in an anaerobic chamber, thus laborious and time‐consuming. Methods and Results: The CAC2634 gene encoding PerR is a known peroxide regulon repressor in Cl. acetobutylicum. CAC2634 in a previously constructed Restriction–Modification system deficient Cl. acetobutylicum mutant SMB009 was disrupted using ClosTron method. The resulted mutant SMB012 can be electrotransformed in air with an efficiency of 1·2–3·1 × 10³ transformants μg^?1 DNA. Conclusions: We demonstrated that the disruption of CAC2634 in Cl. acetobutylicum enables its electrotransformation in air. Significance and Impact of the Study: The electrotransformation process of Cl. acetobutylicum could be significantly simplified, especially when operating multiple electrotransformations.     

Effect of electroporation conditions on cell viability of Frankia EuI1c

The feasibility of electrotransformation of Frankia strain EuI1c was investigated. Cell viability decreased as the capacitance of the electrical pulse increased. The addition of 10% glycerol to the electroporation buffer increased cell viability rates. At a fixed capacitance, cell viability was voltage-dependent and resistance-dependent. Electroporation with a high capacitance was mutagenic and produced antibiotic-resistant cells. Electroporation of Frankia strain EAN1pec chromosomal DNA into strain EuI1c generated several colonies that were resistant to lincomycin or kasugamycin.     

Enhancement of biomolecule transport by electroporation: a review of theory and practical application to transformation of Corynebacterium glutamicum

Selective and reversible permeabilization of the cell wall permeability barrier is the focus for many biotechnological applications. In this article, the basic principles for reversible membrane permeabilization, based on biological, chemical, and physical methods are reviewed. Emphasis is given to electroporation (electropermeabilization) which tends to be the most popular method for membrane permeabilization and for introduction of foreign molecules into the cells. The applications of this method in industrial processes as well as the critical factors and parameters which affect the success of this approach are discussed. The different strategies developed throughout the years for increased transformation efficiencies of the industrially important amino acid-overproducing bacterium Corynebacterium glutamicum, are also summarized.     

电转化法提高平连载体DNA转化效率的研究

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     

Electrotransformation of Thermoanaerobacter ethanolicus JW200

Electrotransformation of Thermoanaerobacter ethanolicus JW200 was achieved using the plasmid, pTE16, and a pUC-based suicide vector, pTEA2. The construct pTE16 is based on the Escherichia coli-Clostridium perfringens shuttle vector pJIR715 and contains a thermostable chloramphenicol (Cm) resistance cassette. Evidence supporting transformation was provided by extracting plasmid pTE16 from presumptive transformants of T. ethanolicus and by PCR specific to the chloramphenicol acetyltransferase (cat) gene on the vector pTEA2. Transformation frequencies of plasmid pTE16 and pTEA2 were 50 ± 7.4 and 30 ± 4.2 transformants per μg plasmid DNA. The results provide the first unequivocal gene transfer method functional in T. ethanolicus.     

德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。     

Bioremediation via In Situ Electrotransformation

Bioremediation of polluted sites relies on bacteria to degrade or transform contaminants into less noxious chemicals. To do so, bacteria require genes that encode the degradation enzymes and the capacity to properly express them, which may be lacking in indigenous bacteria. To increase the ability of indigenous bacteria to bioremediate a contaminated site, this research proposes the use of electrotransformation to facilitate bacterial uptake of exogenous degradation genes. As a proof of concept, a lindane degradation gene (linA) located on a broad host-spectrum expression plasmid (pBLN) was introduced into soil bacteria by electroporation both in vitro, in liquid media, and in situ, in soil. In both cases, the electrotransformed bacteria displayed an increase in lindane degradation and an increase in the linA gene copy number. The use of in situ electrotransformation could improve pollutant degradation rates and could provide another tool for bioremediation.     

Effects of DNA Topology on Transformation Efficiency of Bacillus subtilis ISW1214 by Electroporation

We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonuclease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6±0.6) × 10⁵ transformants per μg DNA higher than that ((2.0±0.3) × 10⁴ transformants per μg DNA) for the cr DNA, using the DNA concentration of 20 μg/ml at an electric field strength of 7kV/cm and a capacitance of 10 μF with a single decayed pulse. The transformation efficiency (TE) for the In DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.