BISC219/F12: RNAi Lab 7

Lab 7: Series 3- Investigating Gene Regulation Using RNAi

In the age of genome sequencing we now know, or can make educated guesses about, the location of every gene in an organism's genome; however, this does not give us any information about the function of the gene product (protein) in the organism. We can use reverse genetic analysis to help us solve this puzzle. There are several tools in the reverse genetics toolbox: directed mutation (point mutations or deletions), overexpression using transgenes, and gene silencing using knockout organisms or double stranded RNA (RNAi). Only RNAi and overexpression have been perfected in C. elegans. Scientists still have not found a way to do in vivo homologous recombination in worms.

We are going to use RNAi as our tool to investigate gene function via reverse genetics. C. elegans is the first animal in which the process of RNAi was discovered. A similar system was identified in plants years earlier but, curiously, that groundbreaking discovery was largely ignored by the scientific community until it was noticed in animal models. We now know that RNA regulation in cells is a fundamental method of regulating gene expression in organisms ranging from microscopic C. elegans to humans. Many labs are now working non-stop to develop treatments for many "incurable" human diseases using RNAi.

In this investigation for Series 3 that you will begin today, each pair of students will be given E. coli bacteria that have been genetically manipulated to contain a vector plasmid that contains the C. elegans lsy-2 gene. The bacteria that contain this plasmid express C. elegans lys-2 double stranded RNA. These bacteria will serve as food for our wild type C. elegans (worms with normal copies of lys-2) and induce the interference RNA pathway in those worms. The result should be a '"knock down" in the amount of mRNA specific to the lsy-2 gene and, thus, a reduction in the amount of lys-2 gene product. We hope to see the effect of this gene silencing in phenotypic differences between wild type worms that were RNAi induced and those that weren't.

Calibration of Micropipettes

Before we begin our molecular genetic experiments, we will practice (AGAIN) properly handling and using the micropipettes.

1. To calibrate your P1000, P200, and P20 micropipets, label 6 microfuge tubes (1-6) and weigh them. Record the weights in the table below.
2. Following the table below, pipet the specified volumes into the pre-weighed microfuge tubes prepared above and then re-weigh them. Record all weights.
3. Calculate the weight of the water in grams. 1000 microliters of water should weigh 1 gram at room temperature.
4. If the water in any tube weighs more or less than 1 gram, ask your instructor for help. If your calibration is significantly off after several repeated attempts, your pipet (or your technique!) may need adjustment.
   

Tube #	Tube Pre-Weight	Vol. in μL using P20	Vol. in μL using P200	Vol. in μL using P1000	Weight of Tube + Water in grams	Weight of Water in grams
1		10	0	990
2		0	100	900
3		20	175	805
4		2	88	910
5		0	200 (5 times)	0
6		20 (5 times)	0	900

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

Picking a Bacterial Colony

Your instructor will give each pair an LB+amp plate on which bacteria containing our pPD129.36 lsy-2 plasmid are selectively growing. Please pick two well isolated colonies, circle them, label one A and the other B and write your initials and group color on the agar side of the plate.

How can you be sure that your colonies contain the plasmid carrying the gene you are interested in studying? In theory, any colony of bacteria growing on your LB+amp plate should contain a plasmid because the gene for antibiotic resistance is not chromosomal, but instead expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. The amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme; therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. You must be careful to pick ONLY the bigger, central colony and not the satellites. The satellite bacteria are unlikely to carry the plasmid that contains the C. elegans lys-2 gene.

This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. Because bacteria reproduce asexually and are immobile on solid media, it is likely that the hundreds of thousands of bacteria making up that colony are genetically identical daughters of a single cell. This allows us to take bacteria from a single colony and sub-culture them in liquid media to make millions of identical copies. However, knowing that the bacteria growing in your broth or on your agar with ampicillin all have the plasmid responsible for amp resistance does not confirm that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that may have a plasmid lacking the gene of interest. That can happen when a vector plasmid is "empty". Our plasmid, pPD129.36 lsy-2, was genetically modified from a purchased base plasmid that was genetically synthesized by a pharmaceutical company. Remember that researchers need to be able to study any gene of interest; therefore, base plasmids are created that allow insertion of any gene of interest in proper alignment with synthetic gene promoters so that the gene of interest can be expressed as desired in a bacterial or a yeast model system. The first step in this process after acquiring a base plasmid such as pPD129.36 is to isolate small segments of chromosomal DNA that contains your gene. Then you must make lots of copies of it by PCR after designing primers that will copy just your gene, and then ligate the gene from the PCR product into the vector plasmid so that it is in proper alignment with an effective synthetic promoter. If all goes well, after transformation (uptake of this plasmid into a cell) the ligation works to achieve this proper alignment but occasionally the ligation fails and the plasmid DNA anneals back on itself. We need a way to find only the colonies with successful ligation that are expressing our gene of interest off the synthetic vector plasmid.

To achieve this goal we are going to do a colony PCR. Instead of adding purified lsy-2 gene fragments as template DNA in a PCR reaction with primers specific for your gene, you will add a TINY little part of a colony as the template for your PCR reaction. During the first heat cycle the cells will burst open and release their DNA into the reaction. We will test both well-isolated, non-satellite colonies per group to be sure we continue with bacteria that have the gene of interest inserted in our plasmid.

1. Obtain three PCR tubes and lids from your instructor in your team color.
2. Label the side of each tube A, B and C - the marker WILL rub off the top. Tube C will serve as your negative control with no colony added.
3. Add 30 ul of master mix to each tube. Your master mix will include: 23 μL H₂O; 3 μL of PCR buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl2 pH 8.3); 0.67 μL of 10 mM dNTPs; 0.67 μL of forward primer (20 μM stock); 0.67 μL of reverse primer (20 μM stock); 2 units/μL Taq Polymerase
4. After each tube has master mix, use the sterile end of an autoclaved toothpick (not the end you are touching) or the end of a sterile micropipet tip and gently touch the center of your colony of interest and pick up a tiny, barely visible amount of the bacteria. DO NOT TAKE THE ENTIRE COLONY!!!
5. Gently twirl the toothpick in the tube or mix the bacteria from the pipet tip with the master mix making SURE that you have gotten it off the tip or toothpick and into the reaction. Discard the toothpick or the tip into your orange autoclave bag.
6. Repeat for colony B
7. Snap the lid on the tubes, pulse them in the microcentrifuge with the appropriate rotor and adapters and bring them to the thermal cycler for PCR initiation.
8. Finally wrap the plate in Parafilm. Store the plate in the refrigerator until the day before the next lab when you will set up an overnight culture.

PCR Conditions:

Step	Temperature	Time	Repeat
1	94°C	2 minutes	1 time
2	94°C	30 seconds
3	54°C	30 seconds
4	72°C	1 minutes	Steps 2-4 30 times total
5	72°C	10 minutes	1 time
6	4°C	forever	end program

Agarose Gel Electrophoresis

After the PCR reactions are completed you will run a gel to analyze the results of the amplification (the search for your gene).
Add 5 μL of loading dye to each PCR reaction.
You will find a precast 1% agarose gel with Sybr-Safe in 1x TAE buffer that is just for your group's use. Make sure that the wells of the gel are closest to the black (negative) electrode and that the gel apparatus has plenty of buffer. Draw a template in your lab notebook so you know which colony is to be put in each lane (1-7 left to right) and which lane contains your DNA ladder. Make sure you get a copy of the DNA ladder key.
Load 15μL of each PCR product into separate wells in the gel. Make sure they are loaded in alphabetical order left to right.
Load 5 μL of the 100 base pair DNA ladder in the well on the far right.

Run the gel for ~ 20min. at ~120 volts.

Capturing Digital Images of Nucleic Acid Gels Stained with SYBR Safe Using the BioRad Imaging System in L308

Instructions for Taking a DNA gel image stained with Sybr Safe using the BioRad ChemiDoc MP System with Image Lab Software

IMPORTANT: Ethidium Bromide stained gels may NOT be imaged in this equipment. Remove gloves and wash hands BEFORE using the computer. DO NOT contaminate the computer. The XcitaBLue Conversion Screen should be kept covering the UV transilluminator and only removed TEMPORARILY when using fluorphores other than Sybr Safe or SybrGreen. Please return the Xcita Blue Screen to the UV transillumintor if you remove it.

Quick and Easy Protocol for photographing Sybr Safe DNA gels:
1) Make sure the Power Button on the right front of the imager shows a green light. If not press it until the green light comes on and wait 5-10 min for warm up.

2) Open the UV transilluminator drawer on the lower front of the imager and make sure that the XcitaBlue Conversion Screen is in place. If not find it and position it covering the glass. Clean it with water and a paper towel if necessary.

3) Position your gel in the center of the glass.

4) Close the drawer, remove gloves, and wash your hands before using the computer to the left of the imager.

5) Open the ImageLab 4.0.1 software by double clicking on the icon on the computer desktop

6) Find and open the Protocol called SybrSafe under Recent Protocols or from the Open Menu. Double click to open it.

7) Click Position Gel (yellow button) to check the position of your gel.

8) Click Run Protocol (green button) to take the photo.

9) To Save your image (if you are not going to analyze it quantitatively), find or make a folder for your work in Documents (NOT on the Desktop). go to File---Export---Export for Publication (use the defaults, e.g. 300dpi). You will see a pop up message reminding you that this option is not optimized for analysis---it doesn’t matter—Click OK. Check the Location where your image will be saved, the FILE Name, and use the drop down menu to SAVE AS TYPE tiff or jpg. Click Save.

10) Close the Image Lab Software 11) Don gloves, remove your gel, clean the glass with water and a papertowel. 12) The computer AND the ChemiDoc Imager should remain ON. DO NOT Turn OFF the power or shut down or log off the computer.

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 that you are sure contains the gene of interest (determined from your visualization of successfully amplified, appropriately sized DNA seen on your gel photo). 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 your LB+amp plate in the glass front refrigerator in a rack labeled with your lab day. Make sure there is some bacteria remaining on the plate of a colony that you saw successful gene amplification in the pcr product.
   
2. Begin by pouring (DO NOT PUT A PIPET INTO THE STOCK LB!!) 10 ml of sterile LB 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.
9. Incubate these broth cultures at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!