Welcome to the Feeding of Worms Page!!!!!

The final step to silence the DPY-10 gene in the N2 worms is to insert the vector (DPY-10 and L4440) that we had previously created into the worms. In order to do this, we need to feed the worms the vector that we created. This is a somewhat difficult process becasue we have to insert a piece of DNA into the bacteria that the N2 worms will eat. In this case, we are going to be feeding the worms HT115. This specific strain allows us to create double stranded RNA which begins the process of silencing the DPY-10 gene. (The double stranded RNA binds to a protein complex, Dicer, which cut the RNA into smaller pieces. Then the RNA pieces bind to another protein complex, RISC, which eliminates one of the strands and binds to the other. The RISC and the single strand bind to mRNA and destroy the mRNA so that the gene is silenced.) HT115 makes the T7 RNA polymerase gene with a lac-operon. The T7 RNA polymerase transcribes RNA in the L4440 vector, creating double stranded DNA and beginning the gene silencing process.

The picture to the right is HT115, showing the T7 RNA polymerase. When the lac operon is induced by IPTG, T7 RNA polymerase binds to the T7 promoters in the picture on the left. The T7 will transcribe RNA in both directions, creating double stranded RNA.

NIcPa1.png NicPag2.png

Making Competent Cells For Transformation
(refer to Transformation page)

Transformation of Competent Cells
  • Add 5 ul of ligated vector (L4440 and Dpy-10) to 100 micro-liters of competent HT115
  • Incubate on ice for 60 minutes
  • Pre-warm AMP plates at 37° C
  • Add 420 ul of LB
  • Incubate at 37° C for one hour
  • Plate 100 ul onto pre-warmed AMP plates

Growing Transformed Cells in a Broth for LB/AMP/IPTG plates
  • Remove a colony from the plates with the transformed cells
  • Place the colony in LB broth with AMP
  • Incubate in shaking water bath at 37° C overnight

Seeding Plates
  • Plate 100-125 ul of the transformed cells (previous step) onto LB/AMP/IPTG plate
  • Incubate at 37° C overnight

Feeding N2 Worms
  • Sterilize a worm pick with alcohol
  • Pick five N2 worms and transfer them to one of the seeded plates (transformed HT115)
  • Repeat this process for however many plates you have
  • Confirm, using the microscope, that the worms are still alive
  • Incubate overnight at 20° C
  • Recheck plates and remove any dead worms

Before and After Pictures
Normal, healthy worms before the DPY-10 gene is silenced.

The would-be worm with the DPY-10 gene silenced if the experiment worked.

Unfortunately as the last day of classes approaches, there is not enough time to finish the experiment. We do not know if in fact the worms will become fat and dumpy. However, the picture provided shows what a N2 worm with the DPY-10 would look like if we had completed the experiment.

Edited by Nicole Page