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

Abstract

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 R 8 Si 8 Mg 6 O 16 (OH) 4 , where R = CH 2 CH 2 NH 2 , 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 β-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3indolyl-β-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (1-4%). 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 (OD 600 = 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 × 10 3 CFU/μg 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.

Spherical and needle shaped magnetic nanoparticles for friction and magnetic stimulated transformation of microorganisms

“…Considering that endocytosis is not performed by E. coli JM109, the form factor of the delivery agent should play an important role in transformation due the requirement of a cell envelope piercing mechanism, which should be promoted by nanoscale needle-like morphologies. In fact, sepiolite and Mg aminoclays, being the usual reported delivery agents capable of promoting transformation of microorganisms, also possess a high aspect ratio between two of their dimensions [74,75], which supports this hypothesis. Moreover, theoretical and experimental studies using high aspect ratio nanomaterials such as vertically aligned carbon nanotubes (CNTs) have suggested that the bending energy stored in the CNTs is a substantial factor for the physical rupture of both Gram-positive and Gram-negative bacteria [76].…”

Application of lamellar nickel hydroxide membrane as a tunable platform for ionic thermoelectric studies

“…In the presence of aminoclay (2 mg mL −1 ) the pH of DI water increased to ∼9.8. Moreover, the FT-IR spectrum of aminoclay possesses a peak at 2053 cm −1 , assigned to the ionized –NH 3 + group 46,47 (Fig. S3b, ESI†).…”

Repurpose the antimicrobial peptide Buforin II for plasmid transformation into Escherichia coli

“…In the investigation on the transformation of plasmid pUC19 into non-competent E. coli JM109, the OD600 of E. coli JM109 and spreading time were studied. The OD600 of 1.0 and spread time of 90 s were favorable for the transformation process (Mendes et al, 2021a). In the study of Mendes et al (2021b), 2-pyrrolidinone-functionalized needleshaped magnetic nanoparticles were used to form the complexes with plasmid pUC19 and the translocation of nanoparticle-pUC19 complexes across the cell envelope of E. coli JM109 were promoted by frictional stimulation, leading to a maximum transformation efficiency of 3.1 × 102 CFU/µg plasmid.…”