This article describes a method for utilizing RNA interference (RNAi) expressing bacteria to inhibit specific gene expression. The protocol outlines the preparation and incubation of these bacteria for further assays.
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 previously prepared RNAi containing bacteria into four milliliters of carbenicillin supplemented LB. Place the plates into a shaking incubator optimized for multi-well plates at 37 degrees Celsius and 950 rpm for 16 hours, then collect the bacteria by centrifugation at 2,000 g for 5 minutes. Decant the supernatant by inverting the plate and vigorously shaking it.
Using 100 microliters of S Basal resuspend the RNAi bacteria. Pipette the resuspended bacteria into an appropriate number of wells of a multi-well NGM plate supplemented with IPTG and carbenicillin and allow the plate to dry.
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