BISC219/F13: RNAi Lab 9

Lab 9: Series 3 - Induction of the Feeding Bacteria to Produce dsRNA and seeding plates for RNAi

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 bacteria containing the plasmid carrying the construct to RNAi the gene that you want to study.

1. Find your plate in the white refrigerator in the back of the lab. Select a large, well isolated colony to start your overnight culture. Do not pick a small "satelite" colony as it may not be transformed with our plasmid.
   
2. Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator.
   
3. Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator) to each tube. Calculate the effective concentration of ampicillin that you have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook.
   
4. Add 5 microliters of the 12.5mg/ml tetracycline stock (also found in the refrigerator). Calculate the effective concentration of tetracycline that you havein your LB broth tube. Record that info in your lab notebook.
5. Gently swirl your LB +amp+tet broth to mix the contents.
   
6. Label the two sterile glass culture tubes with tape in your team color. Label one with hsf-1 feeding bacteria and your initials. Label the other with your initials only.
   
7. Inoculate the broth with your bacteria by using a sterile disposable loop to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony and don’t pick up any satellite colonies! Gently swirl the loop in the LB+amp+tet broth - you should be able to see the colony come off the loop. 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 37C incubator at the front of the room when you come in the door.
9. Incubate these broth cultures at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!
   

To Do Today

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 is very difficult to near impossible 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 an interesting C. elegans gene, three different strains of worms, wild-type, rrf-3 (RNAi enhanced) and CL2070 hsp-16.2::GFP, 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 modified bacteria as dsRNA factories.

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

Done on the night before this lab: You and your partner made an overnight broth culture of your selected colony. This process created a sub-culture of many identical copies of the plasmid carrying the construct that will induce RNAi to silence or downregulate the gene that you want to study.

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 successful induction requires log phase growth.

To create the subculture of bacteria your cultures will be diluted 1:5 (2 mL of culture into 8 ml of LB + amp). These cultures will be allowed to grow for three hours and then induced 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 feeding plasmid and transcribes our C. elegans hsf-1 DNA into RNA!

A simplified map of the C. elegans RNAi plasmid :

More specifically: The bacterial cells of strain HTll5(DE3) contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements. This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene. When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase. This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites. Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell. The IPTG induction allows us to "turn on" and express the plasmid gene of interest only when we want to and it allow us to make much higher levels of RNA for RNA interference than would be made without this induction.

Another useful thing about E. coli strain HT 115(DE3)is that this particular strain is deficient for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect! This E. coli strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transformed cells to grow on media containing ampicillin.

Our goal is to upregulate production of dsRNA of our worm gene of interest, hsf-1 in pPD129.36 plasmids containing that gene in HT115(DE3) bacteria.

To induce your cultures, the lab specialist or your instructor:

1. Added 10 μL of 0.5 M IPTG to 10 ml of a log-phase sub-culture of your cells. What is the effective concentration of IPTG?
2. Put your culture back in the 37°C incubator in the spinning wheel for approximately 3-4 hours.
3. The bacteria worked hard expressing T7 RNA polymerase and transcribing complementary mRNA of C. elegans' hsf-1".

To do after induction is complete:

1. Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.
2. While your cultures are spinning label your 8 "feeding plates" with an F for feeding.
3. When the culture is done spinning, remove all of the supernatant.
4. Add fresh LB to the 2 ml mark on your conical tube.
5. Resuspend the bacterial pellet by gently pipetting up and down with a P1000 pipet set at 750 ul. Use a filter tip found on your bench. This step concentrates your bacteria.
6. In the laminar flow hood in L304 or L301 (labs next door) pipet 200 ul of your induced bacteria onto the center of each feeding plate that you have pre-labeled with an F. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain NGM Lite medium + 50 ug/ml carbenicillin and 0.5 mM IPTG.
7. Allow the bacteria to dry on the surface before you move them.
8. Obtain 8 control plates and label each of them with a C - 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 feeding plasmid is only expressing RNA from the original multiple cloning site region (MCS) of vector - it lacks DNA specific to any worm genes.
9. Wrap your two stacks of plates with elastics and a piece of your color tape.
10. Put both stacks into the large shoebox labeled with your day. The plates will be stored in the 4°C incubator until you are ready to add worms.

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Assignment

Please follow the directions in BISC219/F13:Assignments for completing the graded assignment due at the beginning of Lab 10.