Worm+Feeding!

Home Editor: Sean Lapuk

In this step cultures of the HT115 strain of //E. coli,// which was previously transformed with the L4440 plasmid containing the rol6 insert, are fed //to C. elegans//.

In order to suppress a desired gene, in this case Rol6, with RNAi double stranded RNA needs to infiltrate the nematode's cells. To do so we feed the worms bacteria filled with dsRNA copies of the Rol6 gene. The feeding strain of //E. coli//, HT115, has within its chromosome a gene to create T7 RNA polymerase. The gene comes from T7 viruses, which are able to copy genetic material very quickly if given a T7 promoter to start from. The cloning vector used in the experiment, L4440, has these promoters on either side of our insert allowing both strands of the vector to be quickly and simultaneously copied if T7 RNAp is present. The combination of HT115 and L4440 ensures that we will have billions of dsRNA copies of Rol6 which will initiate RNAi.



Normally, the T7 RNAp gene of HT115 is turned off by the lac operon that comes before it in the sequence. Laci gene codes for a protein that binds to a DNA sequence downstream, preventing RNA polymerases from transcribing RNA from genes downstream of the laci gene. Lactose will bind to the protein, stopping it from binding and allowing normal transcription. IPTG acts simmilarly to lactose and will also block the laci protein. Growing the feeding strain on NGM lite plates with IPTG ensures that T7 RNA polymerases will be transcribed and translated from HT115's chromosome.

In order for HT115 to be of use it must be transformed with the L4440 vector containing the Rol6 gene insert, but HT115 has a low transformation efficiency. DH5alpha //E. coli// are easier to transform, needing fewer plasmids to transform the same number of cells, but lack the T7 RNAp gene. So we use the few L4440 vectors we have to transform DH5alpha and then use the millions of plasmids made by those few transformed cells to transform HT115. By this method we are able to get more HT115 with L4440 than if we had tried to transform them with the original sample of the plasmid.

After growing up the newly transformed culture of HT115 it is a simple matter of spreading the cells onto new NGM lite plates with AMP to select for transformed cell and IPTG to turn on the T7 RNAp gene. Add some nematodes to the plates and let the feast begin. After ingesting their special meal the nematodes should no longer be able to move normally but will instead turn in circles.

Feeding nematodes -spread 300ul of transformed HT115 growing in LB onto 5 warm NGM lite plates with IPTG and AMP -pluck L4 stage (adult) wild type nematodes and put them onto the seeded plates, 2 worms per plate -observe //C.elegans// over the next few days, removing any dead worms from the plates. -Watch the nematodes curl up as their own biological machinery permanently silences their Rol6 gene.



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