TITLE: Transformation Protocol ATMT Trichoderma atroviride
AUTHORS: José Manuel Villalobos-Escobedo

[DESCRIPTION]
This is the protocol for the transformation of conidia by Agrobacterium to perform the BarSeq technique in Trichoderma atroviride, which is reported by Villalobos-Escobedo et al., 2023.

[STEPS]
SECTION: DAY 1:
1. Inoculate 3 plates of Trichoderma @ 27 °C Grow them for 4 days

 

SECTION: DAY 3
3. Make liquid agro induction media (ABI) 

For 100 transformations 5 mL of ABI are needed, water is autoclaved and all others are filtered and sterilized 
200 mL to wash, 200 mL to resuspend (grow)

MIX AND FILTER INSIDE HOOD

 

 

 

 
For AB Buffer check pH, then filter 
SECTION: DAY 3
4. Prepare:  1 mL 3,5-dimethoxy-4-hydroxy-acetophenone (acetosyringone) dissolved in dimethyl sulfoxide (DMSO).  (make this fresh every time)

-- Acetosyringone is filtered through syringe and 0.22 um filter top in DMSO (0.098 g/ mL)
SECTION: DAY 3
5. Autoclave 437ml ddH2O with 20g Bacto Agar
Add MIXED induction liquid to agar.
Mix together inside hood for a few minutes
Pour plates.



Pour solid agro induction media plates and leave on bench to dry for 2 days.
SECTION: Day 4
9. Measure the OD600 of the agro cell culture. (It will likely be around OD600 1.)

(Around 9 am )
SECTION: DAY 3
6. Prepare medium for Agrobacterium LB + Kan.
SECTION: DAY 3
7. Inoculate A. tumefaciens strain harboring the plasmid for Trichoderma transformation onto LB + Kan.250 µL 

SECTION: DAY 3
8. Shake at 250rpm at 30C overnight

(Place around 7 pm- 8 pm)
SECTION: Day 4
10. Back dilute the agro culture to OD600 0.5 in LB+Kan with twice the number of ml of media to the number of transformations you plan to do (i.e., if you are doing 100 transformations back dilute into 200ml LB).

100 mL from stock and 100 mL new LB+Kan
SECTION: Day 4
9.1. In order to get an accurate measurement, dilute the sample 1:10 in H2O in the cuvette.

(100 uL agro cell culture, 900 uL H2O)

SECTION: Day 4
11. Shake at 250rpm at 30C for 2h
SECTION: Day 4
12. Measure the OD600 of the agro cell culture after 2 hrs

 

SECTION: Day 4
13. Centrifuge the agro culture at .4000 rpm, 15 min 

SECTION: Day 4
14. Pour supernatant into waste container.
SECTION: Day 4
15. Wash cells in agro induction media
 

SECTION: Day 4
16. Centrifuge the agro cells at 4000 rpm, 15 min 

SECTION: Day 4
17. Pour supernatant into waste container.
SECTION: Day 4
18. Resuspend cells in agro induction media 



 

18.1. Put cultures on shaker @ 25C and rpm 250, for 24 hrs

SECTION: DAY 5
19. 
 
Approximately 23h after putting agro cells into induction media, collect Trichoderma conidia from plates into 5-10ml of H2O.
SECTION: DAY 5
20. Shake and vortex vigorously to suspend conidia
SECTION: DAY 5
21. Measure conidial concentration

Count four corner squares, and middle square
Avg spores:  Sum of both sides / 10 squares
Math: (Avg. spores)(1x10^4)(25)(100)
SECTION: DAY 5
22. Aliquot 2e7 conidia into 2ml Eppendorf tubes
SECTION: DAY 5
23. Spin cells at 10000 rpm, 5 min 
or 4000 rpm for 20 min
SECTION: DAY 5
24. Pipet off supernatant
SECTION: DAY 5
25. Resuspend cells (pellet) in 2ml of agro induction media cell culture (from yesterday)

SECTION: DAY 5
26. Incubate at room temperature for at least 5 min
SECTION: DAY 5
27. Put a 0.2um sterile PES bottle top filter onto a bottle and set up the vacuum  
 


SECTION: DAY 5
28.  Place a 0.45um HAWP filter onto the bottle top filter using sterile forceps
  
 


SECTION: DAY 5
29. Pipet the 2ml of Trichoderma/agro cell mixture onto 0.45um HAWP filter
 


SECTION: DAY 5
30. Wait for the liquid induction media (ABI) to filter through the bottle top filter
SECTION: DAY 5
31. Using sterile forceps, carefully transfer the filter with cells on it to the solid induction media petri dish, making sure to keep the side with the cells on it facing up! 

SECTION: DAY 5
32. Incubate induction media plates with filters at 20C in the dark for 6 days.  

SECTION: Day 11
33. Label 2 falcon tubes for each HAWP filter 
 


SECTION: Day 11
34. Add 4ml sterile distilled H2O to a 50ml conical tube
 

SECTION: Day 11
35. Using sterile forceps, carefully transfer the HAWP filter with cells on it to the 50 mL conical

SECTION: Day 11
36. 
 

SECTION: Day 11
37. Vortex vigorously to get the rest of the cells off of the HAWP filter
SECTION: Day 11
38. Transfer 2ml of cell suspension to sterile 2 mL Eppendorf tube.

SECTION: Day 11
39. Pipet off supernatant into a waste bottle
SECTION: Day 11
38.1. Spin at10000 rpm, 5 min .

SECTION: Day 11
40. Transfer the rest of the cell suspension to the 2 mL Eppendorf tube and Spin at10000 rpm, 5 min .
Pipet off supernatant into a waste bottle
SECTION: Day 11
41. Resuspend cell mixture into 100 µL sterile distilled H2O

SECTION: Day 11
42. To spread in large plates (150 mm) PDA.


1.- PDA with hyg and Carb (4 ml Hyg and 1 ml Carb per L): dispense 50 ml.
2.- PDA with Carb (1 ml per Liter): dispense 13.5 ml.
3.- Conidia transformants: 1 ml 
4.- Low-melt PDA with Carb (1 ml per L). 13.5 ml. 37 C. we can mix with conidia. 
5.- PDA layer carb and hyg. 13.5 ml, kept at 55 C.



SECTION: Day 11
43. Place at 25C in the light for 7 days (cheking efficiency with Triton)


SECTION: Day 11
42.1. 1 plate with filter goes to 3 plates of top/bottom media
SECTION: Day 11
42.2. 
Plate cells on 
5mL Top: MM + Carb 
25 mL bottom: MM + Carb + Hyg 

You don’t need to dilute the control plate but for the experimental ones:
100%:
1:25:         4 ul of cells in 96 ul of water
 

SECTION: Final Harvest (Day 18)
44.   
 

Dispense 10 mL of sterile water onto the plate


SECTION: Final Harvest (Day 18)
45. Use the sterile glass spreader to detach as many as possible 
SECTION: Final Harvest (Day 18)
46. Transfer into a 50 mL falcon tubes

SECTION: Final Harvest (Day 18)
47. Spin down 50 mL falcon tubes at 4,000 RPM for 20 minutes

SECTION: Final Harvest (Day 18)
46.1. Add another 10 mL of sterile water to plate and transfer into tube 

SECTION: Final Harvest (Day 18)
48. Resuspend pellets in 10 ml of 50% glycerol
SECTION: Final Harvest (Day 18)
48.1. Store in 1.5 mL tubes that are no more than half full, stored at an angle to avoid rupturing the tube.

 

SECTION: DAY 1:
2.  Prepare LB + Kan (500 mL) 

 

18.2. 
 

SECTION: DAY 5
32.1. 
 

SECTION: Day 11
43.1.