Worm+Feeding

// Introduction // There are two primary aspects to worm feeding. The first, feeding the worms so that they stay alive to be used at the conclusion of the lab, is covered in Worm Care and Feeding. The second, feeding the worms “food,” or cells that will then cause them to express blisters under their cuticles is here!

//Background// To silence the genes for the Bli-1 protein, which is responsible for //preventing// blisters from forming on the //C. elegans//, we are utilizing RNA interference, a topic heavily covered here. The vehicle that we have decided to use to induce this gene silencing is HT115 //E. coli// cells. These cells have been transformed with the L4440 vector containing the Bli-1 insert. Thus, they have produced many copies of this L4440 vector, which codes for dsRNA (the result of T7 polymerase sites) which, when digested by the C. elegans, enters the worms’ cells, where it ultimately silences the Bli-1 gene.

//“Where this fits in”// This step is the culmination of weeks of lab work. We are finally taking cells that were transformed by a plasmid (L4440+Bli-1 insert) that was isolated from a previous transformation (of DH5α) that was the result of L4440 being __ligated__ with genomic DNA (of the Bli-1 gene) that was amplified by PCR and isolated from the __genomic DNA of the worms themselves__ and feeding them to the worms, starting the RNAi mechanism.

//Procedure//


 * Transformation of Competent Cells: ** Here we are introducing the L4440 vector with the insert into HT115 cells, so that when we plate these cells, they will produce many copies of the modified vector.


 * Add 5 micro-liters (μl) of ligated vector (L4440 + Bli-1) to 100 μl of competent HT115
 * Incubate on ice for 60 minutes
 * Add 400μl of LB
 * Incubate @ 37°C for one hour
 * Plate 100 μl onto previously pre-warmed AMP plates (37 °C)
 * Incubate overnight


 * Growing Transformed Cells in LB/AMP Broth: ** Here we are ensuring the growth of millions of copies of the colonies of interest by introducing them to a nutrient rich and selective environment


 * Utilizing sterile technique, remove one colony from AMP plates
 * Inoculate LB+AMP broth with colony
 * Incubate in 37°C water bath overnight


 * Seeding Plates: ** Here we are giving the cells a chance to grow on the plate so that when we introduce the worms, they will have special “food” to digest (“food” that contains the modified plasmid and dsRNA).


 * Plate 100-125 μl of the transformed cells from LB+AMP broth onto NGM-lite plate + IPTG + AMP
 * Incubate at 37°C overnight


 * Feeding N2 Worms: ** Here we are introducing the worms to an environment rich with “food” that contains our modified plasmid and dsRNA so that RNAi can do its thang!


 * Utilizing sterile technique, prepare worm pick with alcohol
 * Remove five N2 (adult stage) worms from plates that have been chunked throughout entire lab, and transfer them to one of the seeded plates containing the transformed HT115 cells [cf. Image 1]
 * Repeat for the number of successfully prepared HT115 plates (suggested minimum of four)
 * Confirm that the worms are still alive by looking under the microscope (check for movement)
 * Incubate overnight at 20°C
 * Check plates for dead worms, remove dead worms, and observe phenotype (which has hopefully changed!)

//Conclusion// Unfortunately, we did not get this far in the experiment due to lab errors, inconsistent results, and "stubborn" procedures; however, if given another week, we anticipate that we would have completed the project! We finished after transforming the DH5a cells and creating 11 minpreps (one per successful colony), a topic addressed here.

Image 1 (adult worms to transfer to seeded plates)