Sep 04, 2023
Version 1
Transformation Protocol ATMT Trichoderma atroviride V.1
- José Manuel Villalobos-Escobedo1
- 1UC Berkeley
- ATMT
Protocol Citation: José Manuel Villalobos-Escobedo 2023. Transformation Protocol ATMT Trichoderma atroviride. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbe3plzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 29, 2021
Last Modified: September 04, 2023
Protocol Integer ID: 55461
Keywords: barseq technique in trichoderma atroviride, conidia by agrobacterium, trichoderma atroviride, protocol for the transformation, conidia, agrobacterium, barseq technique, transformation
Abstract
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.
Troubleshooting
DAY 1:
Inoculate 3 plates of Trichoderma @ 27 °C Grow them for 4 days
Note
Sterilize 6 clean flasks
5 bottles
2 100 mL GC
2 250 ml GC
Prepare LB + Kan (500 mL)
Note
50 ug/mL is working concentration for Kan, amount added depends on stock concentration
Ex: 50 mg/mL stock concentration
1 L of LB would require 1 mL of 50 mg/ml Kan
-Usually, I put 1 ml in 500 ml of LB
DAY 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
Note
-- CASamino acids (10% CASamino acids) (Heat up and stir)
-- Glucose (36% Glucose) (Heat up and stir)
--Thiamine (10% Thiamine)
Note
Sterilized bottles
Dataset
ABI Media Spread Sheet
NAME
Dataset
Chemical Mixes for ABI
NAME
For AB Buffer check pH, then filter
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)
- 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.
2d
Prepare medium for Agrobacterium LB + Kan.
Inoculate A. tumefaciens strain harboring the plasmid for Trichoderma transformation onto LB + Kan.250 µL in 50 mL
Shake at 250rpm at 30C overnight
(Place around 7 pm- 8 pm)
Day 4
Measure the OD600 of the agro cell culture. (It will likely be around OD600 1.)
(Around 9 am )
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)
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
Shake at 250rpm at 30C for 2h
2h
Measure the OD600 of the agro cell culture after 2 hrs
Note
It should be around OD600 1. If it is not at least OD600 0.95, put it back on the 30C shaker for another 30min.
Centrifuge the agro culture at .4000 rpm, 00:15:00
15m
Pour supernatant into waste container.
Wash cells in agro induction media
Note
The number of mL should correspond to twice the number of transformations that you are doing
ex: 20 ml for 10 transformations
New flasks are used
Centrifuge the agro cells at 4000 rpm, 00:15:00
15m
Pour supernatant into waste container.
Resuspend cells in agro induction media
Note
The number of mL should correspond to twice the number of transformations that you are doing
For 100 transformations 200 ml of induction media, and each transformation has 4*10^7 conidia,
it's 2*10^7 per ml. Concentration of agro.
We can use tubes with caps for roller.
Put cultures on shaker @ 25C and rpm 250, for 24 hrs
Note
Materials for next day:
-Filter top
- 0.45 um Filter
- Vacum
DAY 5
6d 0h 5m
Note
(using plates of T. atroviride that are 4 days old)
NOTE: Make sure the big square plates are at room temperature.
Approximately 23h after putting agro cells into induction media, collect Trichoderma conidia from plates into 5-10ml of H2O.
Shake and vortex vigorously to suspend conidia
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)
Aliquot 2e7 conidia into 2ml Eppendorf tubes
Spin cells at 10000 rpm, 00:05:00
or 4000 rpm for 20 min
5m
Pipet off supernatant
Resuspend cells (pellet) in 2ml of agro induction media cell culture (from yesterday)
Incubate at room temperature for at least 5 min
Put a 0.2um sterile PES bottle top filter onto a bottle and set up the vacuum
Equipment
Thermo Scientific™ Nalgene™ Rapid-Flow™ Sterile Disposable Bottle Top Filters with PES Membrane
NAME
0.2 μm Bottle Top Filter or equivalent
TYPE
Nalgene
BRAND
09-741-07
SKU
LINK
500 mL, 0.2 μm PES Bottle Top Filter
SPECIFICATIONS
Place a 0.45um HAWP filter onto the bottle top filter using sterile forceps
Equipment
HAWP MF-Millipore Membrane Filter, 0.45 µm pore size
NAME
Membrane filter
TYPE
Millipore
BRAND
HAWP04700
SKU
LINK
0.45 um 47 mm
SPECIFICATIONS
Pipet the 2ml of Trichoderma/agro cell mixture onto 0.45um HAWP filter
Note
Being careful to only get cell mixture on the HAWP filter.
Wait for the liquid induction media (ABI) to filter through the bottle top filter
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!
Incubate induction media plates with filters at 20C in the dark for 6 days.
Dried filters ready for incubation
6d
Note
Materials for next part:
- 50 mL or 500 mL tubes with sterile beads and sterile water
- Layered PDA plates
- MM plates for quantifying
- Sterile water
Day 11
Label 2 falcon tubes for each HAWP filter
Note
25 - 50 filters fit into 500 mL tubes
Add 4ml sterile distilled H2O to a 50ml conical tube
Note
500 mL conical tube
Using sterile forceps, carefully transfer the HAWP filter with cells on it to the 50 mL conical
Note
Use beads and VORTEX until it has all fallen off
Vortex vigorously to get the rest of the cells off of the HAWP filter
Transfer 2ml of cell suspension to sterile 2 mL Eppendorf tube.
Spin at10000 rpm, 00:05:00 .
5m
Pipet off supernatant into a waste bottle
Transfer the rest of the cell suspension to the 2 mL Eppendorf tube and Spin at10000 rpm, 00:05:00 .
Pipet off supernatant into a waste bottle
5m
Resuspend cell mixture into 100 µL sterile distilled H2O
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.
1 plate with filter goes to 3 plates of top/bottom media
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
Note
Make up media in half the amount of water, and autoclave water separately, then combine
Place at 25C in the light for 7 days (cheking efficiency with Triton)
Final Harvest (Day 18)
20m
Washing of plates with water
Note
Materials needed:
- 50% glycerol
- Sterile water
- Glass spreaders
- 1 ml Pipettes
- 25 ml pipettors and pipet tip
- Tips 100-1000 ul
- 15 mL Falcon tubes
Dispense 10 mL of sterile water onto the plate
Use the sterile glass spreader to detach as many as possible
Transfer into a 50 mL falcon tubes
Add another 10 mL of sterile water to plate and transfer into tube
Spin down 50 mL falcon tubes at 4,000 RPM for 20 minutes
20m
Resuspend pellets in 10 ml of 50% glycerol
Store in 1.5 mL tubes that are no more than half full, stored at an angle to avoid rupturing the tube.
Note
Where is it stored? What temperature?