We electroporated 1 μl of each ligation reaction into 19.5 μl of 10-Beta electrocompetent E. coli (NEB, C3020K) with a Bio-Rad E. coli Pulser (2.0 kV, 200 ohms, 25 μF) and 0.1-cm gap cuvettes (Bio-Rad Gene Pulser MicroPulser cuvette, 1652089). Then, we immediately added 980 μl of super optimal medium with catabolic repressor (SOC) medium and incubated the mixture at 37°C with 220 rpm shaking for 1 hour. Then, the electroporated bacteria were plated on LB agar plates with ampicillin at 100 μg/ml using glass beads and incubated overnight at 37°C.
Gene pulser micropulser cuvette
Manufactured by Bio-Rad
Sourced in United States
The Gene Pulser/micropulser Cuvettes are laboratory consumables designed for use with Bio-Rad's Gene Pulser and Micropulser electroporation instruments. They provide a controlled environment for the delivery of electrical pulses to cells or other biological samples during electroporation experiments.
Automatically generated - may contain errors
9 protocols using gene pulser micropulser cuvette
1
Bacterial Transformation by Electroporation
2
Electrocompetent E. coli Transformation
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Transformation of E. coli strains was performed by washing E. coli cells electrocompetent using standard protocols (Wood, 1983 (link)). Basically, 100 ml of overnight culture was harvested during mid-exponential phase (OD600 0.5-0.7), placed on ice for 10 min and centrifuged at 5,000 x g, 4°C for 10 min. The supernatant was poured off and the pellet was resuspended in 100 ml of 10% glycerol. Centrifugation and resuspension steps were repeated twice more with decelerating volumes of resuspension buffer and cells were finally resuspended with 500 μl of 10% glycerol. Transformation of E. coli cells by electroporation was performed in a 0.1 cm cuvette (Gene pulser MicroPulser cuvette) at 1.8 kV (MicroPulser electroporator, Bio-Rad Laboratories).
3
Electroporation and DNase I Treatment of Intestinal EVs
4
Extracellular Vesicle Electroporation and Purification
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The intestinal EV pellet was dissolved in 100 µL PBS. As previously described, these EVs were loaded into a Gene Pulser/micropulser Cuvettes (Bio-Rad) for electroporation (GenePulser Xcell electroporator, Bio-Rad) and then treated with DNase I (300U) for 30 mins, 37 °C.
5
Electroporation of Gut-Derived EVs
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The isolated gut EVs from ageing WT mice were resuspended in sterile PBS. Then, as previously described (23 (link), 50 (link)–52 (link)), these EVs were loaded into a Gene Pulser/micropulser Cuvettes (Bio-Rad) for electroporation (GenePulser Xcell electroporator, Bio-Rad) and treated with DNase I (300U) for 30 mins, 37°C.
6
Electroporation of Extracellular Vesicles
7
Doxorubicin Encapsulation in Extracellular Vesicles
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DOX was loaded into EVs via electroporation using a MicroPulser electroporation device (Bio-Rad, Hercules, CA, USA) and Gene Pulser/MicroPulser Cuvettes (0.1 cm gap; Bio-Rad, Hercules, CA, USA). EVs (20 µg of total EV protein) were mixed with doxorubicin hydrochloride in electroporation buffer (10% glycerol with 500 mM sucrose [34 (link)]; buffer pH was adjusted to 7.0 using sodium hydrochloride). The total mixture volume was 90 μL, and 5 µg/mL doxorubicin hydrochloride for maximal loading of 1 µg EVs (1 µg refers to the total protein amount that was measured by the Bradford assay) was used. EVs were electroporated at 400 V with a double pulse. After electroporation, samples were incubated at 37 °C for 30 min. To remove unincorporated DOX, samples were cleaned using Exosome Spin Columns (MW 3000; Invitrogen, Thermo Fisher Scientific, Bleiswijk, The Netherlands) and filtered 3 times through Amicon 100 kDA filters (Merck, Darmstadt, Germany) (Appendix A , Figure A3 ). The DOX concentration in electroporated samples was calculated according to the standard curve of DOX fluorescence intensity. Electroporated DOX solution without particles in the sample was used as the negative control in further cell viability experiments.
8
Extracellular Vesicle Electroporation and DNA Depletion
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The intestinal EV pellet was dissolved in 100‐µL PBS. As previously described, these EVs were loaded into a Gene Pulser/MicroPulser Cuvettes (Bio‐Rad) for electroporation (GenePulser Xcell electroporator; Bio‐Rad) and then treated with DNase (deoxyribonuclease) I (300 U) for 30 minutes at 37 °C. The depletion of bacterial DNA was confirmed by 16s ribosomal RNA (16s rRNA) quantitative polymerase chain reaction (qPCR) analysis.
9
Complementation of S. oneidensis ΔdegQ Mutant
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The degQ region (SO3942) was amplified from S. oneidensis MR-1 genomic DNA using primer pair degQ-F and R (see Supplementary Table S1). The amplicon was ligated into pHSG399 at the XhoI and BamHI restriction sites to generate recombinant plasmid pHSG399-degQ. The purified pHSG399-degQ (approximately 2 μg) was transformed into ΔdegQ cells by electroporation using 0.2-cm Gene Pulser/MicroPulser cuvettes (Bio-Rad) and a Bio-Rad XPulser. Immediately after transformation, 0.5 mL of super optimal broth with catabolite repression (SOC) medium devoid of antibiotics was added to the cell suspension. Cells were transferred into a sterile culture tube and incubated at 30°C for 1 h with continuous shaking. Following incubation, the cells were spread onto LB agar plates supplemented with 25 μg/mL chloramphenicol and incubated at 30°C for 2-3 days. The complementation strain was named ΔdegQ+degQ.
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