A METHOD FOR PREPARATION OF COMPETENT RALSTONIA EUTROPHA CELLS AND GENE TRANSFORMATION

Background and Aim:Ralstonia eutropha have a broad prospects applying in biodegradation and bioremediation because its ability of produce Polyhydroxyalkanoates (PHAs). The aim of this study was to access a suitable method for preparation R. eutropha competent cells as a key stage for genetically modification and the study of its ability for exogenous transformation.Methods:In this study two factor was conducted to investigate the effects of CaCl2 concentration (20mM, 50mM, and 100mM) and bacterial growth (log-phase levels: OD= 0.2, 0.4, 0.6, and 0.8) in the preparation and transformation efficiency of the R. eutropha competent cells. The pBI121 vector was transformed into prepared R. eutropha competent cell as sample plasmid vector. The accuracy of gene transformation was confirmed by colony PCR.Results:The results showed that concentration of 20 mM CaCl2 and OD: 0.8 are two important factors affecting the transformation efficiency in R. eutropha.Conclusion:The method that we optimized in this study for R. eutropha competent cells preparation is simple and time consuming. Due to R. eutropha is a versatile microorganism and because of biotechnological interest, optimization of a transformation method for introducing exogenous gene, can be used for R. eutropha genome editing and modification of this interest bacteria traits.

Keywords:

Competent cell , Ralstonia eutropha , Gene transformation