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Microfluidic Screening of Electric Fields for Electroporation.

Garcia PA, Ge Z, Moran JL, Buie CR - Sci Rep (2016)

Bottom Line: The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes.Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes.We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Energy and Microsystems Innovation, Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.

ABSTRACT
Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.

No MeSH data available.


Related in: MedlinePlus

Critical electric field (Ecrit) for bacterial electroporation as a function of applied voltage.Panel (a) shows the values obtained from individual experiments, visualizing the data shown in Table 1; panel (b) shows averages (error bars show ± ΔEcrit) for each bacterium at each applied voltage. These values indicate that (gram-negative) E. coli BL21 requires a smaller Ecrit than C. glutamicum and M. smegmatis (gram-positive) bacteria.
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f5: Critical electric field (Ecrit) for bacterial electroporation as a function of applied voltage.Panel (a) shows the values obtained from individual experiments, visualizing the data shown in Table 1; panel (b) shows averages (error bars show ± ΔEcrit) for each bacterium at each applied voltage. These values indicate that (gram-negative) E. coli BL21 requires a smaller Ecrit than C. glutamicum and M. smegmatis (gram-positive) bacteria.

Mentions: Both gram-positive (C. glutamicum and M. smegmatis) and gram-negative (E. coli BL21) strains have been tested in the microfluidic device. Figure 5 plots the electric field thresholds for electroporation of C. glutamicum (5.20 ± 0.20 kV/cm @ 2 kV and 2.5 kV), M. smegmatis (5.56 ± 0.08 kV/cm @ 2 kV and 2.5 kV), and E. coli BL21 (3.65 ± 0.09 kV/cm @ 1.0 kV, 1.5 kV, and 2 kV). The shift of cells during and after the pulse was quantified by tracking the motion of a single fluorescent bacterium at the transition zone and correlating the distance traveled by this bacterium from the last pre-pulse to the first post-pulse image to the corresponding shift in local electric field experienced by that bacterium. The induced shift could be generated by electrophoresis, electroosmotic flow, and/or pressure gradients. This shift in electric field is reported as the uncertainty measurement (ΔEcrit) for each experiment conducted. The average in the thresholds was calculated from the replicates of each strain across all applied voltages. The average calculation across all applied voltages is appropriate since electroporation is an electric-field-dependent physical phenomenon. Consistent with published protocols in the literature, we confirm that E. coli BL21 (gram-negative) requires a weaker electric field to induce electroporation than both gram-positive bacteria studied, suggesting that membrane composition is a potential contributor to the electroporation outcome5.


Microfluidic Screening of Electric Fields for Electroporation.

Garcia PA, Ge Z, Moran JL, Buie CR - Sci Rep (2016)

Critical electric field (Ecrit) for bacterial electroporation as a function of applied voltage.Panel (a) shows the values obtained from individual experiments, visualizing the data shown in Table 1; panel (b) shows averages (error bars show ± ΔEcrit) for each bacterium at each applied voltage. These values indicate that (gram-negative) E. coli BL21 requires a smaller Ecrit than C. glutamicum and M. smegmatis (gram-positive) bacteria.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759816&req=5

f5: Critical electric field (Ecrit) for bacterial electroporation as a function of applied voltage.Panel (a) shows the values obtained from individual experiments, visualizing the data shown in Table 1; panel (b) shows averages (error bars show ± ΔEcrit) for each bacterium at each applied voltage. These values indicate that (gram-negative) E. coli BL21 requires a smaller Ecrit than C. glutamicum and M. smegmatis (gram-positive) bacteria.
Mentions: Both gram-positive (C. glutamicum and M. smegmatis) and gram-negative (E. coli BL21) strains have been tested in the microfluidic device. Figure 5 plots the electric field thresholds for electroporation of C. glutamicum (5.20 ± 0.20 kV/cm @ 2 kV and 2.5 kV), M. smegmatis (5.56 ± 0.08 kV/cm @ 2 kV and 2.5 kV), and E. coli BL21 (3.65 ± 0.09 kV/cm @ 1.0 kV, 1.5 kV, and 2 kV). The shift of cells during and after the pulse was quantified by tracking the motion of a single fluorescent bacterium at the transition zone and correlating the distance traveled by this bacterium from the last pre-pulse to the first post-pulse image to the corresponding shift in local electric field experienced by that bacterium. The induced shift could be generated by electrophoresis, electroosmotic flow, and/or pressure gradients. This shift in electric field is reported as the uncertainty measurement (ΔEcrit) for each experiment conducted. The average in the thresholds was calculated from the replicates of each strain across all applied voltages. The average calculation across all applied voltages is appropriate since electroporation is an electric-field-dependent physical phenomenon. Consistent with published protocols in the literature, we confirm that E. coli BL21 (gram-negative) requires a weaker electric field to induce electroporation than both gram-positive bacteria studied, suggesting that membrane composition is a potential contributor to the electroporation outcome5.

Bottom Line: The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes.Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes.We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Energy and Microsystems Innovation, Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.

ABSTRACT
Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.

No MeSH data available.


Related in: MedlinePlus