Mycobacterial Electroporation
Mycobacterial Electroporation - Related Videos
11:57
Electroporation of Mycobacteria
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Cited by 55 •
2008
Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for...
09:18
Mycobacterial DNA Extraction using Bead Beating in Custom Buffer Followed by NGS Workflow
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2025
This protocol shows bead-beating combined with DNA capture bead purification provides a fast and consistent method for extracting DNA from Mycobacterium tuberculosis samples, making it an effective choice for next-generation sequencing applications.
08:05
Murine In Utero Electroporation
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2023
In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact...
12:19
Bacterial Transformation: Electroporation
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2023
The term “transformation” refers cellular ingestion of foreign DNA. In nature, transformation can occur in certain types of bacteria. In molecular biology, however, transformation is artificially induced through the creation of pores in the bacterial cell walls. Bacterial cells that are able to take up DNA from the environment are called competent cells. Electrocompetent cells can be produced in the laboratory and transformation of these cells can be achieve via the application of an...
07:23
Electroporation of Craniofacial Mesenchyme
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2011
Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.
FAQs
Mycobacteria possess unusually resilient cell envelopes that restrict access to the plasma membrane and make them difficult to transform. Electroporation addresses this barrier by applying a brief, high-voltage pulse that temporarily disrupts membrane integrity. This transient disruption is important because it creates an opportunity for DNA entry without requiring permanent damage to the cell.
The electrical pulse briefly creates pores in the plasma membrane, allowing plasmid DNA or other genetic material to cross into the cell. Because pore formation is temporary, subsequent recovery must support membrane repair and continued cellular survival. The balance between transient permeability and recovery determines whether cells can retain introduced DNA and produce transformants.
Recovery gives electroporated cells time to repair their membranes and resume growth before selection is applied. Selection then enriches for cells that successfully received and retained the introduced genetic material. Keeping these functions conceptually separate helps distinguish cellular repair from identification of transformants, making the resulting population useful for constructing defined genetic strains.
The approach can introduce plasmid DNA and other genetic material into mycobacterial cells. These introduced constructs support several experimental outcomes, including mutants that test gene function, complementation strains that restore or examine genetic activity, and reporter strains that help investigate biological processes. The choice of construct determines the type of genetic question the experiment can address.
A general workflow includes exposing mycobacterial cells to plasmid DNA or another genetic construct, applying a brief high-voltage electrical pulse, allowing the cells to recover under membrane-repair-supporting conditions, and selecting cells with successful transformation. This sequence links DNA delivery to biological recovery and then to enrichment of the genetically modified population.
Researchers use this technique when they need to manipulate mycobacteria genetically to study gene function, virulence, metabolism, or antibiotic resistance. It is especially valuable for organisms that are otherwise difficult to transform because of their resilient cell envelopes. The resulting mutants, complementation strains, and reporter strains provide experimental systems for connecting genetic changes with biological phenotypes.