Development of rapid microwave-mediated and low-temperature bacterial transformations

Abstract

The introduction of exogenous DNA into Escherichia coli is a cornerstone of molecular biology. Herein, we investigate two new mechanisms for bacterial transformation involving either the use of microwave irradiation or a freeze-thaw protocol in liquid nitrogen. Ultimately, both methods afforded successful transfer of plasmid DNA into bacterial cells, with the freeze-thaw technique yielding efficiencies of~10 5 . More importantly, both techniques effectively eliminated the need for the preparation of competent cells.

Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts

“…However, more recent reports claim that a brief freeze in liquid nitrogen for 20 seconds was found to increase the efficiency of freeze-thaw transfection, even obviating the need for standard pre-treatment steps normally employed to make bacteria competent 1256 . Interestingly, microwave irradiation of frozen bacteria/DNA samples was also found to mediate DNA transfection 1256 . Finally, microfluidic reactors have been employed for temperature shock transfection of bacteria 1257 .…”

“…1309 Interestingly, microwave irradiation of frozen bacteria/DNA samples was also found to improve DNA transfection. 1309 Finally, microfluidic reactors have been employed for temperature shock transfection of bacteria. 1310 The advantages include fewer materials, smaller sample volume, and increased precision compared to conventional bulk procedures.…”

“…Mechanistic studies suggest that phase transitions of membrane lipids cause damage to the outer membrane, and are necessary for DNA entry. , Some data indicates that cold shock may not need to go down as low as 0 °C, as the rate and magnitude of temperature changes are more critical than specific temperature extremes . However, more recent reports claim that a brief freeze in liquid nitrogen for 20 s increases the efficiency of freeze–thaw transfection, even obviating the need for standard pretreatment steps normally employed to make bacteria competent . Interestingly, microwave irradiation of frozen bacteria/DNA samples was also found to improve DNA transfection .…”

Effects of Sr2 + on the preparation of Escherchia coli DH5α competent cells and plasmid transformation

“…Thereafter, the chemical transformation methods (Nørgard, Keem & Monahan, 1978;Dagert & Ehrlich, 1979;Zhang, Xu & Xu, 2004) have continuously been modified with various combinations of chemical solutions including different cations (Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ , K + , Na + , Rb + ), PEG, DMSO (Chung & Miller, 1988;Chan et al, 2013) and glycerin Shanehbandi et al (2013). Considering the practicality and convenience of different artificial methods, more efficient exogenous gene transfer systems also have been developed by physical or chemical methods, including chemical procedures (Inoue, Nojima & Okayama, 1990;Sarkar, Choudhuri & Basu, 2002a;Sarkar, Choudhuri & Basu, 2002b;Song et al, 2007), high-voltage electroporation (Dower, Miller & Ragsdale, 1988;Dunny, Lee & LeBlanc, 1991;Sheng, Mancino & Birren, 1995), a biolistic propulsion system (Shark et al, 1991), liposome-mediated DNA transfer (Kawata, Yano & Kojima, 2003), microwaves and ultrasounds assisted DNA transfer (Zarnitsyn & Prausnitz, 2004;Fregel, Rodriguez & Cabrera, 2008;Tripp, Maza & Young, 2013;Deeks et al, 2014) and chemicalphysical (Rb + , sepiolite and nanomaterials) induced transformation (Ren et al, 2017;Ren, Na & Yoo, 2018;Ren et al, 2019), etc. The transformation efficiency (10 4∼10 8 CFU/µg DNA) of different strains can obviously be obtained from these methods described.…”