The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render proteincarrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatinergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the b-amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.

Transformation of non-competent Escherichia coli JM109 was accomplished using pUC19 as donor plasmid and sepiolite as the acicular material to promote cell piercing via application of friction with a polystyrene stick or a magnetic bar on the surface of a hydrogel containing agar. An automatic spreading setup was built with a conventional stirring plate and compared to manual spreading. Several parameters were optimized, namely, the agar content of the hydrogel (2%), concentration of cells (OD=1.3 corresponding to 1.4×10(9) bacterial cells/mL), concentration of sepiolite (0.01%), manual versus mechanical spreading (automatic spreading more consistent) and spreading time (30s). Efficiency values up to 4.1×10(4) CFU/μg pUC19 were obtained. The method proved to be suitable for a rapid and low cost transformation of non-competent E. coli JM109, where higher values of efficiency do not need to be attained.

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 (MNPs) were synthesized by thermal decomposition, functionalized with 2-pyrrolidinone for the attachment of pUC19 plasmidic DNA and used in transformations assays of Escherichia coli JM109. Frictional and magnetic stimulation were employed for promoting the translocation of the nanoparticle-pUC19 complexes across the cell envelope. Transformants were obtained through frictional stimulation using needle shaped MNPs achieving a maximum transformation efficiency of 3.1 × 10 2 CFU/µg pUC19. Magnetic stimulation was also performed using both types of nanoparticles under conventional magnetofection conditions on a magnetic bioreactor and did not induce transformation of E. coli JM109, possibly due to the field intensity at the region of the cells (∼100 mT) not being high enough to overcome the rigidity of the bacterial cell envelope. This work substantiates the need for the delivery agent to have a high aspect ratio in order to achieve transformation of prokaryotes. Moreover, it highlights the limitations of magnetic stimulation for translocation of MNPs across the microbial cell wall, as opposed to magnetofection of eukaryotic cells whereby the entry of genetic material can be readily accomplished using spherical MNPs through an endocytotic uptake mechanism.