Electrical Energy Changes Conductivity and Determines Optimal Electrotransformation Frequency in Gram-Negative Bacteria

Abstract

In many bacterial electrotransformation protocols, pulse time is related to the time constant for a capacitor discharging across a sample of fixed resistance. Using an electroporator which controls pulse time independently of the capacitor time constant, we found that the resistance of bacterial suspensions fluctuates widely during capacitor discharge. With three gram-negative species of bacteria, electrotransformation frequency and survival could be more simply related to the electrical energy delivered in each pulse than to component parameters, such as initial field strength, capacitance, and pulse time. In each case, the number of transformants per survivor increased exponentially and leveled off when more than 0.5 to 1.0 J of electrical energy was delivered. An inverse log-linear relationship between survival and energy delivered was also observed for all three species.

High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain

“…The optimal conditions to obtain P. fluorescens insertional mutants with pUT/mini‐Tn5 Km were defined, first by studying the effect of increasing electric field strengths for a given pulse time [1], and then by studying the effect of increasing pulse times for two different electric field strengths.…”

“…In recent years, transposon‐directed mutagenesis has emerged as a powerful tool to localize new genes in chromosomal locations [1–5]. The main interest of this procedure is to generate non‐leaky mutants in a short time, and to tag the target DNA at the site of mutation.…”