BISC219/F13: RNAi Lab 7

Lab 7: Series 3 - Examining the effect of heat shock on the CL2070 strain

Background materials for HSP's, HSF-1 and the reporter assay are provided by Cassandra Theusch and Peter Kuhn; Elisabeth A. Cox and Theresa M. Grana.

Gene expression is the process in which a gene (DNA) becomes transcribed, translated, and processed to produce the encoded protein and deliver that protein to its proper destination in the cell. Gene expression is the direct result of the Central Dogma of Biology which is the focus of this unit:

(DNA (transcribed into)→ RNA (translated into) → Protein)

In order for transcription of a given gene to take place, a complement of proteins called transcription factors must bind to a unique region of the DNA called a promoter region. In short, in most cases, the binding or release of a protein transcription factor at a DNA promoter region serves to switch gene expression on and off. One way cells respond to stimuli or “stress” is to make or activate specific transcription factors required to bind to a particular gene’s promoter.

Transcription factors interact with → (DNA (transcribed into)→ RNA (translated into) → Protein)

During the next few weeks, you and your group will learn more about the role of heat shock proteins in the stress response. Heat-shock proteins are produced by cells exposed to many different types of stress—not just heat. As their name implies, these widely studied proteins were initially detected when cells were exposed to a transient increase in temperature. This “heat shock” resulted in a temporary loss of cell function, followed by a period of cellular recovery.

In this unit you will study the role of transcription factors and the heat shock proteins they induce during the stress response. Specifically, you will study the induction and activity of heat shock protein 16.2 (HSP 16.2) and a transcription factor called HSF-1 (short for Heat Shock Factor -1) in the model system Caenorhabditis elegans (C. elegans).

During this unit, you will examine the role of of HSP 16.2 and HSF-1 in C. elegans stress response by using green fluorescent reporter proteins (GFP) and phenotypic observations. To help elucidate the level of gene expression and differential cell and tissue expressivity associated with a stress response, you will perform experiments to knock down HSF-1 gene function using RNA interference (RNAi). Your group will investigate how feeding wild-type worms genetically engineered bacteria harboring hsf-1- interfering RNA will alter normal expression of the heat shock factor-1 gene (hsf-1). You will carefully observe the resulting phenotypes of the progeny of these RNAi worms to determine if the RNAi worms’ response to stress differs from control (non-RNAi) worms.

Instructors will do 24 hours before lab:
Your instructor or our lab specialist will come in 24 hours before lab and "heat shock" one plate each of your worms that have been incubating at 15°C and 23°C. The second plate at each temperature serves as your control. Heat shocking involves moving the worms to a 37°C incubator for 30-45 minutes. The worms will then be placed back at their proper temperature until you use them tomorrow.

To Do in Lab Today
We only have one fluorescent compound scope to view the worms and one fluorescent dissecting scope. While some groups are scoring their worms others will be working with the instructor in the microscope room to view and photograph their worms.

1. Bring your 4 plates of worms down to the microscope room.
2. You will first look at your worms and take some pictures under the dissecting scope. Place one of your non-heat shocked plate on the stage of the dissecting scope and expose the worms to the UV light. Record in your notebook what you see. Are the worms glowing? What part of the worms are glowing?

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 one of the colonies of pPD129.36 hsf-1 containing bacteria. The sub-culture you will set up tonight will create many identical copies of bacteria that carry the plasmid containing your gene of interest.

1. Find the LB+amp plate in the glass front refrigerator in a rack labeled with your lab day.
   
2. Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.
   
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 will 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) to each tube. Calculate the effective concentration of tetracycline that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information 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 "pPD129.36 hsf-1" 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 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!