Enhancing DNA electrotransformation efficiency in Escherichia coli DH10B electrocompetent cells

Abstract

Electrotransformation also known as electroporation is the most reliable and efficient tool for plasmid DNA uptake. Electrotransformation efficiency is function of many factors which include (1) number of cell washes prior to electroporation, (2) electroporation cell number, (3) electroporation DNA amount, and (4) cell growth phase. Those factors have limitedly been concomitantly investigated in E. coli DH10B strain. This study is aimed to explore above key factors to define the optimal conditions for high electrotransformation efficiency. The results showed that electrotransformation efficiency of E. coli DH10B was enhanced to 1.5 x 10 9 cfu/µg by washing cells three times with 15 ml of 10% glycerol. This washed off extra salts from cell suspension and enhanced electrotransformation by preventing arcing and enhancing cell resistance while ensuring minimal level of conductivity. Early exponential phase at 0.15 OD600 was the best growth phase for enhancing electrotransformation of E. coli DH10B. The results also showed that higher electrotransformation efficiency was similarly achieved when 0.5 x 10 10 and 0.6 x 10 10 cell numbers were electroporated with DNA amount ranging from 10 to 40 pg. This study confirmed the optimal conditions for electro competent cell preparation and plasmid DNA electrotransformation, which can result highest transformation efficiency.

An Efficient Transformation Method for the Bioplastic‐Producing “Knallgas” Bacterium Ralstonia eutropha H16

“…The phase of cell growth is known to affect transformation efficiency with most transformation methods using competent cells in the early to mid‐logarithmic phase of growth. Different optima had also been suggested . Different phase of cell growth affects both cell number and the ease of transporting DNA across the cell membrane.…”

Optimization of E. coli culture conditions for efficient DNA uptake by electroporation

“…However, efficient growth kinetics of E. coli are not an indication that it will transform efficiently; Wu et al (2010) reported that transformation efficiency is inversely proportional to cell population size. The increased growth rate in the presence of SOC medium might be due to the fact that SOC is a glucose-containing nutrient-rich medium, and glucose is a readily metabolizable sugar (Tu et al, 2005).…”

“…It works by subjecting them to a rapidly changing high-strength electric field. Electroporation is enhanced by several factors; Wu et al (2010) reported that careful control of (i) cell growth phase, (ii) electroporation cell number, (iii) cell desalination with glycerol, and (iv) amount of transforming DNA may enhance transformation efficiency of E. coli up to 10 10 transformants/µg DNA. The combination of chemical-chemical, chemicalphysical, and physical-physical methods improves the transformation manifolds and is used in laboratories worldwide.…”

Construction and optimization of trans‐4‐hydroxy‐L‐proline production recombinant E. coli strain taking the glycerol as carbon source

“…Finally, these expression vectors were tested by restriction enzyme digestion and sequenced. These recombinant plasmids were electro‐transformed into the competent E. coli BL21 (DE3) …”