BISC219/F13: RNAi Lab 8

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(To do on the day before the next lab)
m (Lab 8: Series 3- Creating the feeding strain of bacteria for RNAi in C. elegans)
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In order for RNAi to be successful we must insert the plasmid expressing dsRNA of our ''C. elegans'' gene of interest into the bacteria that will make lots of double stranded RNA.  We will then feed the bacteria that is full of double stranded RNA specific to our gene of interest to our worms.  The process of inserting the plasmid into bacteria is called transformation.  Transformation, either chemically or electrically, opens up pores or channels in the bacterial membrane that allows for uptake of the plasmid DNA.  <br><br>
Precise pipetting is required for transformation to be effective.  To ensure we are all pipetting properly and that our pipettes are properly calibrated follow the procedure below.
Precise pipetting is required for transformation to be effective.  Your instructor will demonstrate the correct technique. If you have any questions, please consult with her/him before proceeding with any micropipetting.
== Part 1:Calibration of Micropipettes ==
== Part 1:Calibration of Micropipettes ==

Revision as of 09:53, 6 November 2013


Lab 8: Heat Shock Proteins (HSP's) and Heat Shock Factor -1 (HSF-1)

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.
Although “heat shock proteins” are best characterized in associated with temperature, mounting evidence suggests that heat shock proteins are expressed and play vital roles during many types of cell stress. Heat shock proteins work by helping other proteins fold properly and have been classified in a functional category of proteins called molecular chaperones. Molecular chaperones catalyze the folding of a newly-translated peptide into the secondary and tertiary structures that characterize the mature protein product. Improperly-folded proteins are unstable, prone to aggregation, and dysfunctional.

Correct protein folding is extremely important for protein and cell function. . Alterations in protein structure, including secondary and tertiary structure, lead to altered protein function. Similarly, improperly or incompletely folded proteins are likely to be dysfunctional and lead to complications for the cell. It should come as no surprise that improper protein folding in cells of the human body can lead to severe diseases. For example, transmissible spongiform encephalopathies (TSE’s), such as mad cow disease, chronic wasting disease, and Creutzfield-Jakob disease, are the result of improperly-folded proteins called prions. A buildup of prions interferes with cell functions and leads to apoptosis (programmed cell death). Other protein folding diseases exhibit similar pathological mechanisms. Alzheimer’s disease is thought to result from the aggregation of improperly-folded amyloid protein. Amyloid plaques accumulate in the brain and damage surrounding neurons as they grow. Certain types of cancer are associated with molecular chaperone dysfunction, which allows tumor cells to grow abnormally and survive adverse conditions.

Heat shock factor 1 (HSF-1) is a transcription factor which is required for transcription/expression of genes encoding many subtypes of heat shock proteins. Under normal cell conditions, HSF-1 expressed in the cytoplasm migrates occasionally, in small amounts, to the nucleus to activate transcription of genes encoding heat shock proteins to carry out normal cell functions. During a cell’s response to stressful conditions, HSF-1 is activated in large amounts, polymerizes, and migrates from the cytoplasm to the nucleus to search out recognized promoter sequences. This high level expression of heat shock proteins allows for refolding of proteins following stress and recovery of protein function. The specific mechanisms and molecular triggers of HSF-1 activation and migration are still unclear. Some evidence points to certain subtypes of heat shock proteins binding with and phosphorylating HSF-1 during the stress response. Yet other HSP’s may have roles inhibiting HSF-1, although scientists are still working to identify all the facets of HSF-1 regulation. More broadly, the types of stressful cellular stimuli which activate HSF-1 also have yet to be elucidated. This is a hot area of research currently, where scientists are hoping to answer some important questions about how cells respond to stressful stimuli. Perhaps your investigations will contribute to this research!

GFP and the reporter assay

A reporter assay is commonly used to assess the activity of transcription factors within a cell. You will use reporter assays to determine functional expression of the transcription factor HSF-1. A reporter assay exploits the expression of a measurable (or visible) protein, in this case GFP. The gene encoding GFP is transgenically engineered to lie downstream of a promoter to which the transcription factor binds and initiates transcription. This engineered “transgene” can be incorporated into the genome of an organism, such as C. elegans. Therefore, when conditions are right and the transcription factor’s expression is high enough, reporter gene is transcribed, and this can be measured by the amount of reporter protein produced.

Green fluorescent protein (GFP) is a commonly-used reporter protein, and, as its name suggests, is a fluorescent protein used in countless ways to visualize and quantify cellular molecules and processes. Originally, GFP was identified in and isolated from the marine jellyfish Aequorea victoria. However, modern advances in molecular biology have produced transgenic forms of GFP to more aptly suit certain experimental requirements. Due to the explosion of discoveries through experiments using GFP, the 2008 Nobel Prize in Chemistry was awarded to Martin Chalfie, Osamu Shimomura, and Roger Tsien for the initial discovery of GFP.

Your experiments will use a transgenic C. elegans strain which has been engineered to contain within its genome a reporter gene. This reporter construct encodes GFP under the control of a hsp-16.2 promoter. (Normally, expression of hsp-16.2 genes results in HSP-16.2 protein production.) HSP-16.2 is one subtype in the family of heat shock proteins, and therefore, it carries out molecular chaperone duties when it is present in the cell. HSF-1 binds to and initiates transcription via the promoter to hsp-16.2. Genetic engineers merely chopped out the promoter to hsp-16.2, pasted it onto the gene encoding GFP, and inserted the combo into a worm’s genome – a reporter gene was born!

Image:Hsp image.jpg

Figure 1: Construction of the GFP reporter transgene. The promoter region of hsp-16.2 was pasted onto the gene encoding GFP. Therefore, transcriptional activity at the hsp promoter can be measured by the levels of GFP in the cell.

Under normal conditions, HSF-1 is present in cells, but its low level of activation cannot be directly measured using the techniques you will use in lab. When a cell is stressed, HSF-1 is phosphorylated and activated in the large amounts measurable using this GFP reporter assay. Activated HSF-1 up-regulates the transcription of the hsp16.2/gfp transgene, which allows measurement of activation through detection of GFP using fluorescence microscopy.




Part 1:Calibration of Micropipettes



200 (5 times)
20 (5 times)

For a Word™ format protocol: Media:Protocol for Micropipet Calibration.doc

To recap what has been done in order for your to be ready to do today's work:
Yesterday you inoculated a few milliliters of LB broth containing E. coli that we are sure have maintained our genetically engineered plasmid, pPD129.36, modified to contain an antibiotic resistance gene to ampicillin and all or part of the C. elegans hsf-1 gene that you want to investigate. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the cells. Overnight the bacteria have grown to high density and the plasmid DNA has undergone many replications. However since you started with a single colony of bacteria and that colony grew from a single transformed cell, all the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another). To isolate the plasmid DNA from this bacterial strain that expresses C. elegans hsf-1 , you will perform what is commonly called a “mini-prep”. This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA so that a certain gene on the plasmid DNA can be studied further.

Synopsis of Today's Work
Today you will transform the isolated plasmid DNA, pPD129.36+hsf-1, into an IPTG inducible strain of E. coli called HT115(DE3), and spread the transformed bacteria onto LB agar media supplemented with ampicillin (resistance conferred by a gene expressed from the plasmid).

Part 2: Plasmid DNA Isolation

What's Happening in Your MiniPrep


Plasmid DNA Isolation Only one of your two overnight cultures should appear cloudy with bacterial growth. If your control is cloudy, please inform your instructor. (You can find the ingredients and concentrations for ingredients of Solutions I, II, and III in the Series 3 Media Recipes page.

  1. To start the plasmid DNA isolation, label 2 microfuge tubes with pPD129.36+hsf-1 (our plasmid name + our C. elegans gene), plus your initials and team color.
  4. If the supernatant is not clear, recentrifuge until it is. When the supernatant is clear, pour it into the waste beaker that is on your bench, then flick the tube with the cap open to remove the last few drops of liquid. The cell pellet will not fall out.

Part 3: Measuring the concentration of plasmid DNA using the NanoDropper

Proper transformation requires the concentration of bacteria to plasmid DNA to be right. We will use a nano-dropper to determine the concentration of the plasmid DNA from a previous plasmid isolation procedure.

Use the ThermoScientific NanoDrop Spectrophotometer in L308 to measure DNA by taking Absorbance at A260nm. This spectrophotometer uses only 1 microliter of sample and does not require cuvettes. The sample is held in place by fiber optic technology and surface tension that holds the sample in place between two optical surfaces that define the pathlength vertically and dynamically. Measurement can be assessed in a range of 2 to 3700nm/microliter dsDNA. These are expensive machines so make sure you follow the directions carefully and ask your instructor for guidance as needed.

More information is available from the manufacturer's website at: |


Using the Nanodropper




4. Perform a blank measurement by loading 1 microliter of purified deionized water.

5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration in your lab notebook and on the tube of plamid DNA. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample.


Part 4: Transformation of plasmid DNA into E. coli strain HT115(DE3)

The competent HT115(DE3) bacterial cells are on the instructor’s bench. You will transform some of your plasmid DNA into these bacteria. The cells are very fragile, so treat them gently.

The genotype of the HT115(DE3) cells is: F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse III minus). This strain grows on LB or 2XYT plates. This strain is tetracycline resistant. The most important things about this strain is:

  1. It has an IPTG inducible T7 polymerase. The chemical IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a lactose mimic and is commonly used to induce the production of large amounts of protein. In this case when the cells are incubated with IPTG lots of the polymerase T7 is made. This T7 polymerase binds to T7 specific promoters on the plasmid and transcribes lots of our gene of interest.
  2. RNAse III minus. The strain is lacking the RNAse III enzyme which typically breaks down double stranded RNAs. We want double stranded RNAs to accumulate.


  1. Obtain a tube containing 50 μL of competent cells from your instructor.
  2. Label the top or the side of the tube with HT115(DE3), pPD12936+hsf-1, and your initials or team color.
  3. Consult with your instructor to determine how much of your plasmid DNA you should add to the cells to start the transformation. When you have determined the correct vol., pipet between 1μL and 10μL of your plasmid DNA to the tube. (The volume you should use is dependent on the conc. you achieved in your mini-prep.) Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit on ice for 10 minutes.
  4. Heat shock by incubating the transformation mix at 42°C for 90 seconds, exactly. This step must be timed exactly. Remove the tube at the end of 90 seconds to your ice bucket while you get your LB ready.
  5. Consult your instructor to determine the volume of warm LB broth you should add (usually between 250-500 microliters) to the transformation mix. LB aliquots should be found in the 37°C mixer/incubator on the green/blue team bench. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip.
  6. Once you have added the LB, close the cap and invert the tube once or twice to mix the contents. Incubate at 37°C for 30-45 minutes in the bench top incubator/ mixer with occasional gentle mixing.
  7. While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label one LB + amp agar plate and one LB + amp + tet plate. Label the bottom of the plated with the strain's identity (HT115(DE3)), the plasmid used, the date, your initials and team color. You must label the bottom of the plated since the tops are easily switched. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.
  8. Once the transformation mix has incubated at 37°C for 30-45 minutes, invert it to mix the contents and pipet 100 microliters of transformed cells onto the center of each of your labeled plates. Pour 5-10 glass beads onto the plates. Put the lid back on and gently swirl the beads all over the agar to spread the transformed bacteria around. When you are done pour the beads into the beaker with disinfectant near the sink.
  9. Replace the lids and leave the agar plates undisturbed for a few minutes to allow for the liquid to be absorbed by the agar.
  10. Once they have dried enough that the surface does not appear wet, invert the plates (lid side down) and incubate them at 37°C for 24 hours. The plates should be incubated with the agar side up so that condensation will not drip onto the surface of the agar and smear the colonies that will be growing there.


Before leaving lab today, give the rest of your isolated plasmid DNA to your instructor in a labeled microfuge tube. Make sure your tube is labeled with your name, lab day, plasmid name, DNA concentration and color coded with a piece of tape in your team color.

To do on the day before the next lab


  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.

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