The fruit fly is an excellent model for molecular genetics. Not only have thousands of genes been characterized in the fly, with regards to their function, we also have resources online that integrate all of the various studies that have been completed in each of these loci. For example, let us say you are interested in immune gene function in Drosophila. You can click on this link here: http://flybase.org/ and search for immune. The result will be 517 hits with that query phrase. Click on “genes” and at the top of the list is the gene imd, or immune deficiency. If we click on the imd symbol it takes us to the gene page, where we can find out lots of information about it, as in “what chromosome is it on?” and “when is it expressed during fly development” or “is it an essential gene?”.
Today we will be asking “Does gene dosage play a role in infection?” by investigating flies from a collection called the Deficiency Kit. These flies each contain a genomic ablation covering a large segment in the chromosome. We will see if these flies have the same number of associated microbes (CFUs) and if they harbor Wolbachia (checked by PCR). As you have already performed these protocols earlier in the semester, this should be a straightforward week!
Isolating DNA from your Flies’’’
One of the big advantages of the molecular revolution in microbial ecology is that we can actually find, based on gene amplification, the presence of Wolbachia in your samples. In order to do that, we will follow the protocol below which first involves liberating the DNA in your sample, then amplifying a Wolbachia specific gene.
Isolate Genomic DNA From Your Sample
Please wear gloves during this protocol
1. Your instructor will provide each of you with three different flies. One is your “unknown” deficiency strain, one is infected with Wolbachia and one is uninfected. Obtain these three flies in 1.5 mL tubes from the instructor bench.
2. To each of the tubes, add 200 uL of PBS and 20 uL of proteinase K.
3. Using a sterile pestle, grind the fly sample in the tube as well as you can. Throw the pestle in the biohazardous waste when you are done.
4. Add 200 uL of buffer ATL to each 1.5 mL tube and mix by vortexing.
5. Incubate your mixture at 56C for 10 minutes (the water bath in the back).
6. Once the incubation is done, add 200 ul of ethanol (100%) to the suspension and mix by vortexing.
7. For each tube, label an individual DNeasy mini spin column in a 2mL collection tube.
8. Pipet the entire mixture into the appropriately labeled column.
9. Spin the column at 6,000 g for 1 minute. Your DNA will now have adhered to the column. Discard the flow through and place the column in a new 2 mL collection tube.
- the next steps are all washes *
10. Add 500 ul AW1 to the column. Centrifuge for 1 minute at 6,000 g. Discard the flow through and place the column in a new 2 mL collection tube It is extremely important that you place the column into a new tube as you will carry over the washes at each step otherwise.
11. Add 500 ul AW2 to the column and centrifuge for 3 minutes at max speed. Discard the flow through and place the column in a labled, 1.5 mL tube
12. Elute the DNA from the column by adding 200 ul of buffer AE to the center of the spin column. Incubate the column for 1 minute at room temperature and spin at max speed for 1 minute.
Your DNA is now in the liquid that came through the column!
Finding the Wild Wolbachia
We will next attempt to amplify only Wolbachia by using a primer set specific to the bacetrium :a forward primer, wsp_F1: GTCCAATARSTGATGARGAAAC , and a reverse primer, wsp_R1: CYGCACCAAYAGYRCTRTAAA. These primers are short sequences of single stranded DNA that are complementary in sequence to areas of the wsp gene in the Wolbachia genome. The wsp gene encodes for a protein on the surface of Wolbachia. After 30 cycles of polymerase chain reaction in a thermal cycler, the result will be a pcr product containing hundreds, if not thousands, of the wsp gene, but only if your fly was infected with the bacterium.
Protocol for PCR
1. Obtain one 0.2ml pcr tube from your instructor - you will need one for each of your DNA extractions (for each of your fly "types”, so three total). All of the ingredients listed below in the table, except the template DNA, have been added together previously and kept on ice for you in these tubes.
2. Label the tube with a fine tipped Sharpie on the side - make sure you keep track of the code name in your lab notebook. Do not use tape!
3. To each tube, you will add 4 μL of the DNA you extracted. Since your pcr tube already has 10μL master mix, 4μL DNAase free water, and 1μL of each of 2 primers, the total reaction volume for everyone will be 20μL.
4. Dispense the template DNA into the liquid directly, watching to make sure that the liquid has left the pipette tip.
5. Bring your tube to your instructor; they will show you where the thermal cycler is located in JH 022. Keep track of where in the PCR machine your tubes have been placed (the exact quadrant, row and column). Your instructor will start the reaction when everyone's tubes are loaded.
| Component || amt. in a 20 μl|
| Final Conc.
| 4 μL already in tube.|
Want to achieve
total of 20 μl reaction vol.
Add from 0 - 3μl
| 2x Phusion Master Mix
|| 10 μl
| wspF primer
|| 0.5 μMolar
| wspR primer
|| 0.5 μMolar
| template DNA
|| 4 μl
|| optimum is 100ng of DNA/reaction
The cycling program is shown below.
94°C for 2 min
37 cycles at:
94°C for 30 s
59°C for 45 s
72°C for 1 min 30 s
1 cycle at:
72°C for 10 min
After the PCR reactions are complete, your instructor will save your reactions for next time. On Thursday, you will run your PCR products on a gel to determine if you have amplified Wolbachia.
‘’’Plating for CFUs: how many microbes do this flies harbor?’’’
Again, obtain the same three flies from your instructor (make sure you have the same deficiency stock you used in the DNA extraction protocol!). Your job is to first homogenize the flies using a sterile pestle and 200 ul of sterile water (this will be your original fly lysate) and then create a dilution series in the same. You will then plate the dilutions.
1. For each fly lysate, remove two sterile 1.5 mL tubes at your bench (in the plastic containers with lids).
2. Label these tubes 1 and 2.
3. Add 0.9 mL of ddH20 to each of these tubes "what micropipette will you use?".
4. Add 0.1 mL of your original fly lysate to tube 1. Cap the tube and mix by vortexing.
5. Add 0.1 mL of the liquid in tube 1 to the liquid in tube 2. Cap tube 2 and mix by vortexing.
6. At this point you are ready to plate your dilutions. For each fly, you will label two solid agar plates (MRS or LB, you will plate on both) with your initials and the dilution factor. "make sure you label the agar side!".
7. Uncap your agar plate. Take 200 ul of your first dilution (tube 1) and spot it onto the center of the agar. Using sterile glass beads, spread the liquid as evenly as possible across the surface of the agar.
8. Replace the cap and allow your sample to sit, undisturbed, agar side down, for ~2 minutes.
9. Repeat the plating and spreading for dilution tube 2 and for each of the two media types.
At the end of this exercise you will have four total plates - two dilutions on both MRS and LB - for each fly sample. For example, as you are working with 3 different types of flies, you will have 3 x 4 = 12 total plates at the end of this exercise.
At the end of this lab remember to:
1) Put your DNA in the -20C freezer
2) Wipe down your lab bench with the 70% ethanol spray bottles and paper towel
3) Place your agar plates in the incubator.
4) Wash your hands!