Lab 7 Fluoresence microscopy and protein localization; continuation of Neurospora genetics

What's learned
1.Fungal genetic stock center website (www. fgsc. net). The stock center contains all experimental fungal strains. Knockout lines of certain genes can be ordered from the stock center. For example,it provides all information for Neurospora.

2. Knock out a fungal gene: homologous recombination.
This method can directly replace the coding region to block the gene function completely. It has advantage over point mutation.

Objective:
1.Pick single ascospore from mating plate for heat shock.

2.Observe nuclei, actin, tubulin, Mak-2 under fluorescence microspores.

3.Observe mushroom/Chlorophylum sp. basidiospores, Buller's Drop and hyphae clamp connection.
The purpose for this exercise is to practice on picking single ascospores from agar block, and taking video for Neurospora hyphae growth. Additionally,to get a general idea of some subcellular structure localization.

Material and Method:
Material:4% Water agar plate;picker,scaple,slides
Method:
1.Picking random ascospores

• Use scaple to cut water agar plate into 4cm block and put it on slide
• Spread loopful of spores-not too many because we want it well seperate
• Flame picker briefly-the advantage of the picker is it can cool down immediately.
• Use the picker as scaple to cut out a block with spores
• Pick up block of agar by scoping`0.2-0.3 mm square
• Transfer to agar tubes
• Transfer rack of tubes to refrigerator to allow spores to hydrate.
• Heat shock:Next day incubate the rack of tubes at 65 degree water bath for 45min.

2. Observer fluorescence signal from Nuerospora mutant.

• Cut a block of agar which is a little smaller than slide with hyphae tip in the middle. Gently lay the agar block on the slide and lay a long cover slide on the top without push. 
• Observe fluorescence signal under microscope. 

Result:
1. 10 ascospores are picked from the water agar block and transfer to slide medium agar tubes. Tubes are incubated at room temperature.

2. Observe microtubel tagged with GFP in SMRP11 mutant and nuclei tagged with GFP in SMRP10 mutant.
Under microscope I observe hyphae grow every second. The spizenkoper is wandering
because the hyphae is looking for nutrient source. Time lap is setup to capture each second of hyphae growth.

3. Mushroom/Chlorophylum sp. morphology observation:
Cut gill and observe basidispores.

4. Cut carp to observe hyphae and clamp connection.

Discussion:

From this experiment, I learned how to recognize ascospores and how to use a picker to pick every single ascospore. I also observe basidiospores and clamp connection from mushrooms. The clamp connection observation give me a vivid image of what clamp connection look like in natural condition which help me to understand the lecture better. I think we should do this kind of experiment more. After a lecture of some typical fungal morphogenesis, we are provided a chance to observe what it actually look like other than just get impression from textbook. It will help us to understand the lecture better and leave a longer time memory on the knowledge.