Synergy h1m

All experiments were performed in E. coli TG1 cells [F′ traD36 lacIq Δ(lacZ) M15 pro A⁺B⁺/supE Δ(hsdM-mcrB)5 (r_k⁻ m_k⁻ McrB-) thi Δ(lac-proAB)]. Plasmid combinations were transformed into chemically competent TG1 cells, plated on LB + Agar plates containing 100 µg/mL carbenicillin and 34 µg/mL chloramphenicol, and incubated overnight ∼17 h at 37°C. The plates were taken out of the incubator in the morning and left at room temperature for ∼7 h at which point three colonies were picked to inoculate 300 µL of LB containing carbenicillin and chloramphenicol at the concentrations above in a 2-mL 96-well block (Costar 3960). Cultures were grown overnight, ∼17 h at 37°C at 1000 rpm in a Labnet Vortemp 56 benchtop shaker. Four microliters of this overnight culture were then added to 196 µL (1:50 dilution) of supplemented M9 minimal media (1×M9 minimal salts, 1 mM thiamine hydrochloride, 0.4% glycerol, 0.2% casamino acids, 2 mM MgSO₄, 0.1 mM CaCl₂) containing the antibiotics above and grown for 3 h at the same conditions as the overnight culture. One hundred microliters of this culture were then transferred to a 96-well plate (Costar 3631) containing 100 µL of phosphate buffered saline (PBS). Fluorescence (FL) (485 nm excitation, 520 nm emission) and optical density (OD 600 nm) were measured using a Biotek Synergy H1M plate reader.

Electrical fields were applied vertically and horizontally to PEMs to investigate their effects on destabilizing PEMs. For the vertically electrical field treatment, conductive PPy films were used as working electrodes. After filling PBS into the wells without bubbles, stainless steel was placed on the top of the wells as counter electrodes (Figure 1c). Regarding the horizontally electrical field treatment, electrodes were placed at two opposing ends of the conductive PPy films (Figure 1d).
After electrical field pretreatment, the dissolutions of DNA and PEI from PEMs in PBS were evaluated at different time points. Because amines of PEI can react with TNBSA to form chromogenic products, 20 μL of TNBSA (0.01 vol% in sodium bicarbonate buffer, pH 8.5) was added to 40 μL of samples [27 (link)]. After 1.5 h incubation at 37 °C, 20 μL of 10% SDS aqueous solution and 10 μL of 1 N HCl was added to terminate the reaction. The absorbance at 335 nm was determined by spectrometry (Synergy H1m, Biotek, Winooski, VT, USA), which were compared to the calibration curve determined by the results of standard PEI solutions to calculate the release of PEI. For DNA evaluation, samples were directly measured at the absorbance of 260 nm because PEI does not contribute any absorption at this wavelength [14 (link)].