Electroporation-Based Delivery of Fluorescent Biomolecules into Bacterial Cells

Begin with a chilled cuvette containing a suspension of electrocompetent bacterial cells, which have increased susceptibility to membrane permeabilization.

The suspension also contains biomolecules tagged with organic fluorophores.

Apply a high-voltage electric pulse to create temporary membrane pores, enabling biomolecule entry into the cytoplasm.

Add a nutrient-rich medium and transfer the cells to a tube. Incubate to promote membrane repair.

Centrifuge the cells, then discard the supernatant containing non-internalized biomolecules.

Resuspend the cells in a high-salt buffer containing a detergent to detach surface-adhered biomolecules.

Centrifuge again, discard the supernatant, and resuspend the cells in the buffer.

Transfer the suspension onto a filter and centrifuge to discard the flow-through containing any non-internalized biomolecules. Then, rinse the cells with fresh buffer and resuspend them.

Mount the cells onto an agarose pad and place a coverslip. The fluorescently labeled bacteria are ready for visualization.

Starting with a stock of fluorescent biomolecules prepared in a low ionic strength buffer, transfer up to five microliters of the biomolecules into either a 20 microliter