A critical combination of operating parameters can significantly increase the electrotransformation efficiency of a gram-positive Dietzia strain.

Shelian Lu, Yong Nie, Yue-Qin Tang, Guangming Xiong, Xiao-Lei Wu

Journal: Journal of microbiological methods 2015;103():144-51

PMID: 24892513

Full Text on Elsevier Science View on PubMed

Abstract

Dietzia spp. have broad potential applications in industries. However, genetic manipulation of these species is obstructed by their low transformation efficiency, which is in the range of 10(4)colony-forming units (CFU)μg(-1) exogenous DNA. In this study, both single-factor and orthogonal experiments were conducted to test the effects of competent cell concentration (parameter A), electroporation time (B), and field strength (C), as well as the concentrations of glycine (D), isonicotinic acid hydrazide (INH, E), Tween 80 (F), and penicillin G (G) on the electrotransformation efficiency of Dietzia sp. DQ12-45-1b. The order in which the parameters contributed to electrotransformation efficiency was C>D>F>G>B>A>E, suggesting that field strength, glycine, and Tween 80 each had a greater capability to increase electrotransformation efficiency than the other parameters tested. Using the optimized protocol deduced from the results of the orthogonal experiments, the electrotransformation efficiency of Dietzia sp. DQ12-45-1b reached 2.51×10(7)CFUμg(-1) plasmid. To the best of our knowledge, this is the highest transformation efficiency that has been reported for Dietzia bacteria to date. Thus, we present a method that combines the transformation factors to obtain the optimal transformation efficiency for a target bacterium.

Address: Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100871, PR China.; College of Architecture and Environment, Sichuan University, Chengdu 610065, PR China.; Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School, Campus Kiel, Kiel 24105, Schleswig-Holstein, Germany.; Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100871, PR China. Electronic address: [email protected].

DOI: 10.1016/j.mimet.2014.05.015