Cramer Lab Aspergillus Protoplast Transformation Protocol
Aspergillus fumigatus Protoplast Transformation
Day 1
This is a checklist of items that should be prepared before you begin a transformation.
- DNA for transformation, either circular plasmid, linearized plasmid or PCR product (read below to see how much is used per transformation, respectively). Best results are obtained with PCR products!
- GMM + 0.5% yeast extract + appropriate supplements (if auxotroph, i.e. UU) in liquid broth.
- 2-3 day old GMM agar plates with your A. fumigatus strain streaked out
- Harvest conidia from 4 petri plates with 25 ml of 0.05% Tween 80
- Rub plates to remove all conidia.
- Harvest first plate, then take harvested solution and transfer to second plate and use that solution for further harvesting. This way the solution gets progressively darker over the 4 plates.
- Transfer final 20 ml into a 50 ml conical tube using a sterile miracloth filter to remove debris, should be dark green (about 10^8 spores/ml).
- SMM plates for plating protoplasts onto (make fresh, day before or day of to avoid contamination in the fridge).
- Top Agar SMM 0.7%
- The rest of the required materials and solutions (Lysing Enzymes from Sigma, Osmotic Media, Trapping Buffer, STC, PEG, Calcium Chloride, autoclaved tubes)
1. Pour about the 10-12 mls of conidia suspension into 250 ml of GMM + 0.5% YE + appropriate supplements for strain you are transforming. The media should turn light green, NOT dark green. 4 plates is almost always enough if they are 3 days old.
2. Here the protocol is variable but CRITICAL. Depending on your schedule, you can increase the temperature and decrease the time, or vice versa, but this is been what’s been the most successful. Incubate at 300 RPM, 28 C for 10 hours. Generally we put the culture in at 10pm and it is done at 8am. Time depends on strain. Less growth is better than overgrowth. You DO NOT want mycelia to form. You just want the spores to germinate and have germ tubes 2-5 times the diameter of the spores themselves. This process is very fickle and if you can’t achieve the correct stage of germination with these exact time points and temps, experiment with a combination of 12-16 hours, 24-28 C. This is VERY strain dependent. We use a shaker in a cold room that is set to a timer to avoid timing issues.
Day 2
1. Take 10 ul of O/N culture and check germination with microscope. Again, looking for germ tubes, NOT mycelia. If no germ tubes present, put back in shaker and check every half hour until ready. KEY: NO MYCELIA
2. When culture is ready, let settle on your lab bench at an angle for 5 minutes, the germinated spores should settle down at the bottom of the flask nicely, almost like a carpet. If you don’t get settling they are not ready!
3. Pour off as much media as possible, leaving ~100 mls behind, without disturbing the germlings. Swirl remaining media distributing germlings and pour into 2 50 ml conical tubes (FALCON). Collect germlings by centrifuging at 3000 RPM for 8 minutes, pour off as much media as possible leaving germling pellet.
4. While centrifuge is going: Add 10 ml of Osmotic Medium to 4 50 ml conicals.
5. Add 35 mg of Lysing Enzyme from Sigma to each conical tube and gently mix until dissolved.
6. Add fungal tissue to conical tubes using a spatula. Add 2 to 3 scoops per tube. KEY POINT Tissue must be COMPLETELY re-suspended, NO CLUMPS! Use the serological pipette. Be violent if you have to. Usually at least 15-20 times up and down to turn the chunks of tissue into a very fine suspension. The amount of tissue is KEY to a good protoplast yield. You want your final solution to be a nice light green in color. Too dark or too light and the digestion will not work. This is an art, sorry, ….. but it works!
7. Gently shake to mix enzyme in. Incubate at 50 RPM 28 C. Digestion time varies, but 5 hours is ideal. Anything less could lead to decreased protplast yields. Too long and cell walls start to reform!
8. Transfer digestions to autoclaved Kimble glass tubes for centrifuging. Be gentle. Overlay 5 ml of Trapping buffer 1 ml at a time using a pipetter. You are creating an interface between the OM buffer fungal digestion culture and trapping buffer. This is an important step! Lean the tube at a 45 degree angle and let the trapping buffer SLOWLY trickle down the sides of the glass tube. Do not disrupt the OM buffer layer!
9. Centrifuge 15 minutes, 5000 RPM, using the sealed swinging bucket rotor.
Day 2 (Cont)
10. At this point you will have several layers in your tube (hopefully). At the bottom will be spores and undigested material, towards the middle will be partially digested tissue and right above this should be a THIN CLOUDY layer...these are your protoplasts. If you do not see a cloudy layer you can remove the top trapping buffer and re-digest the tissue for additional time. This often can work, but if you’ve digested for five hours then you probably aren’t going to improve the situation. Honestly, even if you do NOT see a nice white cloudy layer, take 1 ml of the interface and proceed ….. sometimes the protoplasts get trapped in the partially digested material .. use the microscope, check, I guarantee you will have protoplasts almost every time ….. IT IS OKAY IF YOUR PROTOPLASTS ARE NOT 100% PURE!!!
11. Use the large pipetter (1 ml tips) to pull off only the cloudy layer and put into a 15 ml conical ON ICE. Generally you pull off about 1 ml per tube to give you about four mls of protos into one conical. This will be used for a single transformation. IF you get good yields in all 4 conicals, you can do 2 transformations. KEEP PROTOPLASTS ON ICE FROM HERE ON OUT.
12. GENTLY add cold STC to about 12-14 mls of the 15 conical tube. (This should be 3X the volume of protoplasts that you added.)
13. Centrifuge 6,000 RPM for 8 minutes. Can used fixed rotor for this.
14. Now you should see a small whitish strip along the bottom/side of the tube. These are your protoplasts. Pour off supernatant carefully. Re-suspend protoplasts in 1 ml of STC. KEEP ON ICE.
15. Transfer 1 ml to 1.7 ml microcentrifuge tube and spin at 13,000 RPM for 30 seconds. Should have nice pellet at bottom.
16. Re-suspend protoplasts in 120ul of STC. Can check and count at this point with microscope.
17. Add your DNA to ~100ul of protoplasts. Take 20 ul of protoplasts as control (no DNA). For DNA, I add 10 ug of plasmid, or 10 ug of PCR product. You want the volume to be minimal though. Generally, strive to add no more than 20 ul of DNA and IDEALLY ADD NO MORE THEN 10 ul of DNA solution. Incubate on ICE for 50 minutes. (This is a good time to melt TOP AGAR and get SMM plates out to warm to room temperature).
18. Transfer DNA + protoplast into culture tube and add 1.25ml of PEG solution. Mix WELL by tapping tube. (PEG solution = 950 ul PEG 60% + 50 ul 1M CaCl2. For 1.25 ml mix 1900ul of PEG and 100ul of CaCl2.) Incubate at room temperature for 20 minutes.
19. Add STC to the 4 ml line in the culture tube. This allows ease of plating.
20. Plate onto selective media. I generally plate about 300 ul of solution per plate, meaning 12-15 SMM plates. Add 300 ul to middle of plate and then add 5 ml warm top agar 0.7% and swirl plate. Have to work quickly here, the top agar should be warm and close to solidifying! Also, prepare two NO DNA plates that will serves as the (–) control. You may want to do one plate with just SMM and top agar to make sure that any sort of contamination isn’t the result of the media. Parafilm and incubate right-side up O/N at 37 C.
Day 3
1. Transformants will appear typically appear between 36-48 hours. You do not want colonies to grow that much. As soon as you can see a colony, pick it! Check several times the following day and pick colonies that arise before 48 hours. If the colonies start to sporulate, you will get cross contamination.
2. Using a toothpick, stab outside edge of colony and then stab the middle of a small plate. Let colony grow for 2-3 days so it begins to sporulate and you can prepare glycerol stocks and cultures for DNA analysis etc.
3. Analyze mutants with PCR, Southern blots, etc.
Modified from Nancy Keller’s Lab at UW-Madison – Thank you!
By Robert Cramer Jr.
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