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African Journal of Biotechnology

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Optimization of plasmid electrotransformation into Escherichia coli using Taguchi statistical method

Mohamad Heiat, Hossein Aghamollaei, Seeyed Mostafa Hoseinei, Reza Abbasi Larki, Kheirollah Yari

Abstract


Electroporation is a mechanical method used to introduce polar molecules into a host cell through the cell membrane. In this procedure, a large electric pulse temporarily disturbs the phospholipid bilayer allowing molecules like DNA to pass into the cell. Application of statistical methods to determine the appropriate processes have been suggested for genetic engineering and biotechnology technique such as electroporation. This study explains the use of Taguchi statistical method to optimize the conditions for efficient plasmid transformation into Escherichia coli via electroporation. In order to improve electroporation, optical density of bacteria, recovery time and electrical parameter (field strength and capacitance) were optimized using the Taguchi statistical method. ANOVA of obtained data indicated that the optimal conditions of electrotransformation of pET-28a (+) plasmid into Escherichia coli BL21(DE3)pLysS was 0.7, 120 min, 12 kV/cm and 50 μF, for optical density of cell culture,  recovery time, field strength and capacitance, respectively. The most significant alterations are decrease in field strength and increase in optical density in comparison with common electroporation protocol. The maximum level of plasmid transformation obtained under optimal condition was 8.7×108 transformants/μg DNA plasmid, which was 6.7 fold higher than the control condition.

Keywords: Electroporation, Taguchi statistical method, Plasmid




http://dx.doi.org/10.5897/AJB11.4078
AJOL African Journals Online