Culturing and Maintenance of RNAi-Expressing Bacteria

Begin with a plate containing pre-grown RNA interference- or RNAi-expressing bacteria.

These bacteria carry plasmids containing an antibiotic resistance gene and a target gene insert that produces double-stranded RNA or dsRNA to inhibit specific gene expression in the host.

Using a sterile pin, transfer the colonies into a deep-well plate containing an antibiotic-supplemented medium.

Incubate the plate with shaking for uniform bacterial growth, where the deep wells provide aeration.

The antibiotic eliminates bacteria that lack the plasmid, ensuring that only plasmid-bearing bacteria survive.

Centrifuge the plate to separate the bacteria, and discard the supernatant.

Resuspend the bacteria in a buffered saline solution to stabilize them.

Dispense the resuspended bacteria into a multi-well plate containing nutrient-rich medium supplemented with both an antibiotic and an inducer.

The antibiotic selects for plasmid-bearing bacteria, while the inducer triggers dsRNA expression.

The plate seeded with RNAi-expressing bacteria is ready for further assays.

To test multiple RNAi bacterial strains in parallel in a single well of a 24 well deep well plate, inoculate a single colony from each clone of pr