BISC 219/F10: RNAi Lab 9

Lab 9: Induction of HT115(DE3) feeding strains for RNAi

Through reverse genetics we will deduce the function of a gene starting with its sequence and working back to its phenotype. There are many genes in the genome whose phenotype when mutated is lethal; therefore, it's impossible (or very difficult) to tie function to a particular gene in the traditional forward genetics manner of creating random mutations, looking for phenotype changes, and then finding the defective gene responsible for that function.

In our reverse genetics study of some interesting C. elegans genes, two different strains of worms, wild-type and rrf-3 (RNAi enhanced), are fed bacteria expressing dsRNA specific to a particular worm gene. Ingesting dsRNA initiates cascade of events that leads to the destruction of the mRNA of the target gene. An altered phenotype in the progeny of RNAi-treated worms indicates what happens when the normal function of this gene is lost or significantly downregulated.

Double stranded RNA (dsRNA) can be introduced to the C. elegans cells in many different ways including: feeding, injection and soaking. Each of these methods has positives and negatives. We are using the feeding method - where we use genetically modififed bacteria as dsRNA factories.

To begin to investigate the power of reverese genetics, you will need to grow your own induced bacteria to seed your plates for RNAi feeding.

To do on the day before the next lab: You and your partner will return to the lab to make an overnight broth culture of your selected colony as described below. This process will create a sub-culture of many identical copies of the plasmid carrying the construct to RNAi the gene that you want to study.

1. Find your plates in the glass front refrigerator in a rack labeled with your lab day. You and your partner may reassess your choice of the one well-isolated, medium to large colony, selected previously. You may change your selection, if needed. After agreeing upon an appropriate colony, start your overnight culture.
   
2. Begin by pouring (DO NOT PUT A PIPET INTO THE STOCK LB!!) 10 ml of sterile LB +tetracycline broth from one of the stock containers in the refrigerator into a sterile orange-capped 15ml conical tube. You will use the volumetric marks on the tube for measuring the media rather than using a pipet. Make sure the LB stock does not look cloudy (indicating contamination by a previous user) and take care not to contaminate it yourself.
   
3. Add 10 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator). Calculate the effective concentration of ampicillin that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook.
   
4. Replace the cap of your LB +amp broth and invert the tube several times to mix the contents.
   
5. Label two sterile glass culture tubes (found in a rack in the lab) with tape in your team color. Label one with "pL4440 and the gene name" and your initials. Label the other with your initials only.
   
6. Using a 5 or 10 ml sterile disposable pipet, pipet 4 ml of your working solution of LB+ampicillin broth into each of the 2 tubes. Be careful not to touch the tip to anything non-sterile.
   
7. Inoculate the broth with your bacteria by using a sterile toothpick to scrape your candidate colony off the plate. Be sure not to touch the plate with the toothpick except on the desired colony and don’t pick up any satellite colonies. Make sure the toothpick falls into the sterile broth. The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.
   
8. Balance the 2 tubes across from each other on the rotating wheel in the incubator at the front of the room when you come in the door and incubate them at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!
   

On the morning of lab:
Your instructor or the lab staff will come in early in the morning and sub-culture your bacterial overnight. The cells will be in stationary phase in the morning and sucessful induction requires log phase growth.

To create the subculture of bacteria your cultures will be diluted 1:10 (500 μL of culture into 4.5 ml of LB + amp + tet). These cultures will be allowed to grow until lab time - approximately 3-4 hours.

When you come in to lab you will induce your cultures to make lots of dsRNA by adding IPTG to the culture and letting it continue to incubate for a few hours so the cell is full of dsRNA. The IPTG will compete with the repressors on the lac o promoter and remove them and allow the gene for T7 RNA polymerase to be transcribed and then translated into the RNA polymerase protein. The T7 RNA polymerase then binds to the T7 promoters on the pL4440 plasmid and transcribes our C. elegans DNA into RNA!

To induce your cultures:

1. Add 5 μL of 0.5 M IPTG to your culture. What is the effective concentration of IPTG in your culture?
2. Put your culture back in the 37°C incubator in the spinning wheel for approximately 3 hours.
3. After the lab introduction, we will head up to the penthouse to discuss the papers you were assigned.

To do after induction is complete:

1. Pour your culture into a 15 ml orange cap centrifuge tube.
2. Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.
3. Remove 3.5 ml of the supernatant.
4. Resuspend the bacterial pellet in the remaining 1.0 ml of supernatant - you are concentrating your bacteria.
5. Pipet a 200 μL aliquot of your induced bacteria onto the center of 4 feeding plates. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin and 12.5 μg/mL tetracycline.
6. Allow the bacteria to be absorbed into the media
7. Obtain 2 control plates - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, except that the pL4440 plasmid is only expressing RNA from the vector - it lacks DNA specific to any worm genes.
8. Stack all 6 plates and wrap with an elastic - put them in the lab box with a piece of your tape on top.
9. We will allow the bacteria to continue to induce overnight at room temperature.

4 days before next lab:

1. Come into lab and find your stack of plates.
2. On 2 of the experimental plates add 2 L4 wild type (N2) hermaphrodites
3. On 2 of the experimental plates add 2 L4 rrf-3 hermaphrodites
4. On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites
5. On 1 of the control plates add 2 L4 rrf-3 hermaphrodites
6. Wrap all of your plates in an elastic and stick in your lab day box in the worm incubator set at 23°C
   

You will score your phenotypes in the next lab.