EFFECT OF NEWLY SYNTHESIZED POLYMERS ON GENE TRANSFORMATION IN ESCHERICHIA COLI

Abstract

Transformation is a process used widely in molecular biology research and biotechnology applications for gene cloning, protein expression, and genetic engineering. However, transformation efficiency is generally low and influenced by many factors, which must be improved to enhance cloning technology and reduce the associated time, effort, and expense. Recent studies have shown that many factors can affect transformation efficiency including ionic species, plasmid concentration, ice bath time, and the recovery treatment time. Recently, cationic polymer has been recognized as an effective transformation enhancer in increasing transformation efficiency. In this study, we assessed the potential of two cationic poly(β-amino ester) polymers, which were synthesized through an aza-Michael addition reaction between piperazine and isosorbide diacrylate, as well as poly(ethylene glycol) diacrylate. These polymers were investigated for their ability to enhance DNA transformation. Preparing competent cells from E.coli strain DH5α was optimized by using the Ca2+ and adding Mg2+ ions. To do it, different concentrations of the polymers were applied in pRSET EmGFP before transforming into competent cells through the heat shock method. The colonies on the Ampicillin-added LB (Luria Bertani) agar were analyzed and performed PCR reaction then the transformation efficiency was calculated. Based on our findings, we observed a successful transformation of the emGFP gene, and the two cationic poly(β-amino ester) polymers at high concentrations (20 - 100 ng/ul) increased transformation efficiency more than twice as compared to normal transformation. Notably, the polymer derived from isosorbide had the highest transformation efficiency at a concentration of 20 ng/μl, while the poly(ethylene glycol)-based polymer had lower transformation efficiency at low concentrations than the former.