Experiment #1: Make protoplasts with aspergillus oryzae

To gain momentum and familiarize myself with the technique of making fungal protoplasts, I am going to replicate a protocol from this paper, using a different buffer and lytic enzymes.

• Made MEA + Kan (autoclaved) & YPD + Kanamycin plates in microwave 65/L50g YPD Broth15g Agar 0.05g Kanamycin

• Transferred spores & mycelia from dried Koji plate, 2 each media

 

Mycelia on different media. YPD shows to be the better media for this species.

With fresh cultures, it's time to start the digestion.

• Made a snailase stock solution which is reported to solubilize at 200mg/mL *** At first, I tried adding the mg to the buffer solution — NOT GOOD IDEA, it froths and does not solubilize easily. 

Buffers + Enzyme solutions:

0.5 M KCl + 1.5% enzymatic solution

4.625mLmL buffer

375uL snailaise stock solution 

pH: 7, added 10ul hydrochloric acid for 5

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0.6 M MgSO4 + 1.5% enzymatic solution

4.625mL buffer

375uL snailaise stock solution 

pH:7, added 10ul hydrochloric acid for 5

*Store at room temp, use promptly

Added 700mg lallzyme to 4.625mL KCl buffer, inverted and lightly agitated to dissolve.

Then, added 375ul of 200mg/mL snailase

pH: 7, added 10ul hydrochloric acid f05 5pH Made more solutions with snailase + lallzyme

1.5% snailase 

.7g/5mL lallzyme

Protocol:

• Syringe filter solutions  into new 15mL tubes (lost 2-3mL in the process, very difficult to filter)

• Syringe filtered 10mL of DI water, pipetted 1mL in each 10mL tube

• Used an inoculation loop to grab fungal tissue and suspend in 1mL sterile  DI water

*** Bad idea, very hard to get tissue and each sample will be different. In the future, use liquid culture

 

Mixed 1mL fungal liquid suspension with 1mL of enzyme/buffer solution as written about here.

• Digested at 80rpm, 30°C incubation for 2 hours 

• Control: 2mL DI water of liquid culture mix. Pipetted small amount before incubation to look at under the scope

	0.6 M MgSO4 1.5% Snailase	0.5 M KCl 1.5% snailase 14% lallzyme	Distilled water
Before digestion
After Digestion		2x	2x

Round 2:

Redoing to protocol with liquid culture. Liquid culture was grown in malt + kanamycin and put on an orbital shaker for 3 days. 400+ mL was near fully grown at the time of digestion.

• First make more snailase stock solution. I will use everything I have.

710mg snailase suspended in 3550ul DI water. Did not vortex mix. Just waited. 

• Make 0.5 M MgSO4 buffer + Lallzyme ~ 5mL

• • 616mg added to 15mL tube 

  • 700mg lallzyme added to 15mL tube

  • 4.625mL DI water added to 15mL tube 

  • Inverted until dissolved

  • Checked pH = 6/6.5 → added 10ul hydrochloric acid to 4.5/5

• Added 375ul snailase stock solution for a final read of:

0.5 M MgSO4

1.5% snailase 

14% lallzyme

pH: 5

*** checked pH again after adding snailase. Seems like a solid 5. The pH of pure snailase stock solution was 7/7.5.

• Syringe filtered. And found a much better solution! Use smaller syringes AND smaller filters, diameter wise. Filtering was pretty smooth in 3mL syringe with dime sized .22um filter. Tried with a larger size .22um filter and it still sucked/ the liquid escaped to behind the plunger.  

• Solution was labeled and left on the bench to start digestion tomorrow morning.