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 introducing DNA into mycobacterial cells.
Total time
~3-5 days (including cell recovery and antibiotic selection)
Model organism
Mycobacterium smegmatis
Steps
1
Prepare electrocompetent mycobacterial cells
Grow and wash mycobacterial cells in appropriate growth media, then resuspend in low-conductivity buffer to reduce electrical resistance during electroporation. This preparation is critical for efficient DNA uptake.
▶ 01:30
2
Perform electroporation of M. smegmatis
Apply brief high-voltage electrical impulses to electrocompetent cells mixed with plasmid DNA in a cuvette. This creates temporary pores in the mycobacterial cell wall allowing DNA entry.
▶ 05:34
3
Recover and select transformed colonies
Allow electroporated cells to recover in growth media without selection pressure, then plate on selective media containing appropriate antibiotics to identify successfully transformed mycobacterial clones.
▶ 11:34