RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli
Trascrizione
– In C. elegans, RNAi can be induced by feeding worms bacteria that express double-stranded RNA or dsRNA from a recombinant DNA plasmid.
To begin, grow tetracycline-resistant HT115 E. coli transfected with L4440 plasmid overnight on selective LB agar plates containing tetracycline and ampicillin, or its functional analog carbenicillin. In addition a the target DNA, the plasmid contains a gene for ampicillin resistance. Only bacteria colonies that inherit the plasmid can grow di the presence of these antibiotics.
Next, harvest bacteria colonies and expand them di liquid LB medium a the desired concentration. To induce dsRNA expression, transfer the bacteria solution onto prepared nematode growth medium, or NGM, agar plates containing IPTG.
In this expression system, IPTG mimics lactose a inactivate the lac repressor enabling the expression of T7 RNA polymerase. On the plasmid, target DNA expression is regulated by two convergent T7 promoters. Consequently, sense and anti-sense sequences are transcribed leading a the expression of dsRNA.
Finally, the worm uses the sequence information from the ingested dsRNA a downregulate endogenous mRNAs with complementary sequences. In this experiment, we will prepare RNAi plates with transgenic E. coli for C. elegans feeding.
– For the RNAi plates, load 6-centimeter plates with NGM agar containing IPTG and carbenicillin. Let them dry for 24 a 48 hours at room temperature di the dark. Then, transfer them a 4 degrees Celsius for up a 14 days.
Next, streak selective plates containing carbenicillin and tetracycline with RNAi bacteria clones with L4440 plasmid carrying the lin-53 gene. Use a three-phase streaking pattern and be sure a plate an empty vector control. Then, grow the plates overnight at 37 degrees Celsius.
The next day, pick at least three single colonies and inoculate each of them into a separate culture tube with 2 milliliters of LB supplemented with carbenicillin, but not tetracycline. Plan a have three healthy cultures per condition.
Next, grow the cultures overnight at 37 degrees Celsius until they reach an optical density at 600 nanometers of 0.6 a 0.8. Then, add 500 microliters of each bacterial culture a a 6-centimeter NGM agar RNAi plate. Incubate the plates overnight at room temperature di the dark.
