BISC 219/F10: RNAi Lab 7

Lab 7: Series 3- Reverse Genetics: Picking Your Transformant

Colony PCR
In theory, any colony of bacteria growing on your LB+amp plate should contain a vector plasmid because the gene for antibiotic resistance is not chromosomal, but 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. However, 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.

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, if you have an isolated colony on a plate, it is likely that the hundreds of thousands of bacteria making up that colony are daughters of a single cell (genetically identical). This allows us to take bacteria from a 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 plate with ampicillin all have the vector plasmid responsible for amp resistance, that does not mean that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that have a plasmid without the gene of interest. That can happen when, during ligation, the plasmid DNA annealed back on itself. We need a way to find the colonies that are expressing our gene of interest off the vector plasmid.

To achieve this goal we are going to do a "colony PCR". Instead of adding purified bli-1 or rol-5 gene fragments as template DNA in a pcr reaction with primers specific for your gene (as we did in LAB5), 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 7 well-isolated, non-satellite colonies per group to be sure we continue with bacteria that have the gene of interest inserted in our plasmid vector.

1. Obtain a strip of PCR tubes and lids from your instructor. DO NOT separate the tubes!
2. Label the side of each tube 1-7
3. Find 7 medium to large size, well isolated colonies and circle them and number them 1-7.
4. 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
5. After each tube has master mix, use the sterile end of an autoclaved toothpick (not the end you are touching) and gently touch the center of your colony of interest - starting with #1 - and pick up a tiny, barely visible amount of the bacteria. DO NOT TAKE THE ENTIRE COLONY!!!
6. Gently twirl the toothpick in the tube then discard the toothpick in your orange autoclave bag.
7. Repeat until all 7 colonies are picked.
8. 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.

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 tube.
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 numerical order 1-7 left to right.
Load 5 μL of the 100base pair DNA ladder in the well on the far right.

Run the gel for ~ 20min. at ~120 volts.
Directions for using the Kodak imager to photograph your gel can be downloaded at: Media: How_to_take_gel_photos.doc

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 vector plasmid containing your gene of interest.

1. Find your transformation 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!
   

Outline of Experimental Design for REVERSE Genetics Project

Where are you now in this process?(What have you done so far; What's next?)
Make the feeder strain of bacteria

1. Amplify gene of interest by pcr ;
   
2. Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
   
3. Clean up DNA (remove enzymes);
   
4. Cloning: ligate gene into vector plasmid with amp resistance gene ;
   
5. Transform competent bacterial cells;
6. Select for transformants on media with ampicillin;
   
7. Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
8. Culture the selected colony from colony pcr to create a lot of copies of these bacteria
9. Isolate the cloned plasmid DNA from that cultured colony by miniprep;
   
10. Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
    
11. Select for transformants on media with ampicillin
12. Choose an isolated colony to culture and make lots of feeder strain bacteria;
    
13. Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction.
    
14. Seed NM lite worm growth media plates with feeder strain produced as described
    

Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).

Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.

Isolate RNA from RNAi worms and control worms of same strains.

Perform RT-PCR (Reverse Transcriptase)to amplify the C. elegans gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.

Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.

Assignment

Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.