RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli
Transcrição
– 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 para the target DNA, the plasmid contains a gene for ampicillin resistance. Only bacteria colonies that inherit the plasmid can grow de the presence of these antibiotics.
Next, harvest bacteria colonies and expand them de liquid LB medium para 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 para 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 para the expression of dsRNA.
Finally, the worm uses the sequence information from the ingested dsRNA para 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 para 48 hours at room temperature de the dark. Then, transfer them para 4 degrees Celsius for up para 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 para 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 para 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 para 0.8. Then, add 500 microliters of each bacterial culture para a 6-centimeter NGM agar RNAi plate. Incubate the plates overnight at room temperature de the dark.
