Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

• Autor(a) principal: Mendes, Gabriel Pinto
• Data de Publicação: 2021
• Outros Autores: Kluskens, Leon, Lanceros-Méndez, S., Mota, M.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/1822/71045
• topic: Magnesium aminoclays; Membrane permeation; Microorganism transformation; Nanobiohybrid complex; Plasmid delivery; Science & Technology

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R8Si8Mg6O16(OH)4, where R = CH2CH2NH2, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl ?-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (14%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD600 = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 103 CFU/?g pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109.