Influences of plasmid forms and electric pulses on transformation efficiency in yeast using electroporation

Paper Details

Research Paper 01/04/2018
Views (318) Download (32)
current_issue_feature_image
publication_file

Influences of plasmid forms and electric pulses on transformation efficiency in yeast using electroporation

Riaz Ahmed, Niran Aeksiri, Pongsanat Pongcharoen, Kawee Sujipuli
Int. J. Biosci.12( 4), 188-195, April 2018.
Certificate: IJB 2018 [Generate Certificate]

Abstract

The electroporation systems was widely used for transforming eukaryotic gene. The purpose of this research was to compare effects of plasmid forms and electric pulses on transformation efficiency in yeast using various electroporation approaches. The pPICZαA-crypt plasmid, containing the beta-cryptogein gene, was digested and undigested by SacI to produce linearized- and circular-plasmid, respectively. Both plasmid forms were transformed into competent cells of Pichia pastoris (Y11430) using either electric pulseof 1.5 or 2.0 KV. The single colony was selected and cultured in YPD medium, containing 100 µg/ml zeocinTM, and successful transformants were confirmed by using PCR-amplification.The result showed that the linearized plasmid in both electric pulses significantly generated higher transformants (average 29.25-30.00 colonies/plate) than the circular plasmid (7.63-8.13 colonies/plate). Twelve putative transformants in each transformation system were assessed successful transformation efficiency through PCR with present of the inserted crypt gene and AOXI. The result showed that transformation efficiency of the linearized plasmid (12 of 12 transformants) was significantly higher (p=0.01) than the circular plasmid (10 of 12 transformants) in both electric pulses. This study indicated that the linearized plasmid might be used for increasing electroporated transformation efficiency in P. pastoris.

VIEWS 16

Amelot N, Carrouche A, Danoun S, Bourque S, Haiech J, Pugin A, Ranjeva R, GrimaPettenati J, Mazars C, Briere C. 2011. Cryptogein a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium‐dependent manner. PlantCell andEnvironment 34(1), 149-161. http://dx.doi.org/10.1111/j.1365-3040.2010.02233.x.

Ang RP, Teoh LS, Chan MK, Miswan N, Khoo BY. 2016. Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichiapastoris. Electronic Journal of Biotechnology 19(3), 9-17. http://dx.doi.org/10.1016/j.ejbt.2016.01.007.

Bessa D, Pereira F, MoreiraR, Johansson B, Queirós O. 2012. Improved gap repair cloning in yeast: treatment of the gapped vector with Taq DNA polymerase avoids vector self‐ligation. Yeast29(10), 419-423. http://dx.doi.org/10.1002/yea.29.19.

Calmels T, Parriche M, Durand H, Tiraby G. 1991. High efficiency transformation of Tolypocladiumgeodesconidiospores to phleomycin resistance. Current Genetics 20(4), 309-314.

Cebrián J, Castán A,  Martínez V, Kadomatsu MJ, Parra C, Fernández-Nestosa C, Schaerer, Hernández P, Krimer D,  Schvartzman JB. 2015. Direct evidence for the formation of precatenanes during DNA replication. Journal of Biological Chemistry 290(22), 13725-13735.

David F, Siewers V. 2015. Advances in yeast genome engineering. FEMS Yeast Research 15(1), 1-14. http://dx.doi.org/10.1111/1567-1364.12200.

Dobeš P,Kmuníček J, Mikeš V, Damborský J. 2004. Binding of fatty acids to beta-cryptogein quantitative structure−activity relationships and design of selective protein mutants.Journal of Chemical Information and Computer Sciences 44(6), 2126-2132. http://dx.doi.org/10.1021/ci049832x.

Lau YL, Fong MY. 2008. Toxoplasma gondii: Serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichiapastoris. Experimental Parasitology 119(3), 373-378. http://dx.doi.org/10.1016/j.exppara.2008.03.016.

Lõoke M, Kristjuhan K, Kristjuhan A. 2011. Extraction of genomic DNA from yeasts for PCR-based applications.Biotechniques50(5), 325-328. http://dx.doi.org/10.2144/00.0113672.

Michal J, O’Donohue, Gousseau H, Jean CH, David T, Jean CP. 1995.Chemical synthesis, expression and mutagenesis of a gene encoding beta-cryptogein, an elicitin produced by Phytophthoracryptogea. Plant Molecular Biology 27(3), 577-586.

O’Donohue MJ, Boissy G, Huet JC, Nespoulous C, Brunie S, Pernollet JC. 1996. Overexpression in Pichiapastoris and crystallization of an elicitor protein secreted by the phytopathogenic fungus, Phytophthoracryptogea. Protein Expression and Purification 8(2), 254-261. http://dx.doi.org/10.1006/prep.1996.0098.

Oswald N. 2007.E.colielectroporation vs chemical transformation.Retrieved 19-02, 2018, from https://bitesizebio.com/10297/ecoli-electroporation-vs-chemical-transformation.

Sanchez O, Aguirre J. 1996. Efficient transformation of Aspergillusnidulans by electroporation of germinated conidia. Fungal Genetics Reports 43(1), 48-51. http://dx.doi.org/10.4148/1941-4765.1317.

Schifferdecker AJ, Siurkus J, Andersen MR, Joerck-Ramberg D, Ling Z, Zhou N, Blevins JE, Sibirny AA, Piškur J, Ishchuk OP. 2016. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkerabruxellensis yeast. Applied Microbiology and Biotechnology100(7), 3219-3231. http://dx.doi.org/10.1007/s00253-015-7266-x.

Tercé-Laforgue T, Huet JC, Pernollet JC.1992. Biosynthesis and secretion of cryptogein, a protein elicitor secreted by Phytophthoracryptogea. Plant Physiology 98(3), 936-941. http://dx.doi.org/10.1104/pp.98.3.936.

Version E, Carlsbad C. 1999. Multi-copy Pichia Expression Kit, USA: Invitrogen.

Wang JH, Hung W, Tsai SH. 2011. High efficiency transformation by electroporation of Yarrowialipolytica. The Journal of Micrology49(3), 469-472. http://dx.doi.org/10.1007/s12275-011-0433-6.

Weidner M, Taupp M, Hallam SJ. 2010. Expression of recombinant proteins in the methylotrophicyeastPichiapastoris. Journal of Visualized Experiments (36), 1862. http://dx.doi.org/10.3791/1862.

Wu S, Letchworth GJ. 2004. High efficiency transformation by electroporation of Pichiapastoris pre-treated with lithium acetate and dithiothreitol. Biotechniques 36(1), 152-154.