Journal of Microbiology

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High Efficiency Transformation by Electroporation of Yarrowia lipolytica: Jia-Hung Wang , Wenpin Hung , Shu-Hsien Tsai; J. Microbiol. 2011;49(3):469-472. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0433-6

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Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of Y. lipolytica, we report here that pretreatment of Y. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 1010/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform Y. lipolytica that will be able to yield an efficiency of 2.1×104 transformants/μg for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for Y. lipolytica transformation.

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Optimum Conditions for Transformation of Synechocystis sp. PCC 6803: Xiaonan Zang , Bin Liu , Shunmei Liu , K.K.I.U. Arunakumara , Xuecheng Zhang; J. Microbiol. 2007;45(3):241-245.; DOI: https://doi.org/2536 [pii]

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Abstract: This study was conducted to determine the optimal conditions for introduction of exogenous DNA into Synechocystis sp. PCC 6803. Of the three transformation techniques studied, electroporation, ultrasonic transformation and natural transformation, natural transformation showed the highest efficiency. Additionally, this study demonstrated that the higher plasmid concentration and longer homologous recombining fragments resulted in a greater number of transformants. For successful transformation, the lowest concentration of plasmid was 0.02 μg/ml, and the shortest homologous recombining fragment was 0.2 kb. Use of Synechocystis sp. PCC 6803 in the logarithmic growth phase resulted in two-fold higher transformation rate than that of the same organism when cells in the latent phase or the plateau phase were used for transformation. Pretreatment of the host strain, Synechocystis sp. PCC 6803, with EDTA (2 mM) for two days prior to transformation increased the transformation efficiency by 23%. Additionally, incubation of the cells and DNA for 5 h under light conditions increased the transformation efficiency by two orders of magnitude. Moreover, recovery treatment of the cells before they were plated onto antibiotic medium also increased the transformation efficiency.

Optimal condition for efficient DNA transfer in filamentous cyanobacteria by electroporation: Poo , Ha Ryoung; J. Microbiol. 1997;35(3):181-187.

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Abstract: 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 revealed 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 concentration of DNA; even small amounts of DNA (0.01 ㎍/ml) were able to give a large number of transformants (1.0 × 10³ cfu/ml).

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