Testing Agrobacterium on Fungal Hyphae

February 13: Testing AMT RUBY 35s Plasmid in Filamentous Fungi

This experiment is to see if 1. Our agrobacterium can infiltration fungal hyphae with replicated infiltration media from referenced paper and 2. if 35s plasmid system is expressed.

Materials

Selection plates:

1 - MEA + Hygromycin 50ug/mL (1mL of stock) + 50ug/mL kan(100ul of stock) + 100ug/mL Amp (100ul of stock) (3.5g MEA, 100ul h2o)

2 - IM Agar + hygromycin + 50ug/mL kan + 100ug/mL Amp (3.5g agar + 80mg dextrose 10mM) 

Co-culture plates:

3 - MEA + vanillin (3.5g MEA, 100ul h2o) + vanillin solution

4 - IM Agar from paper (1.5g agar + 80mg dextrose 10mM)

5 - IM Agar from our kits (1.5g agar + infiltration media)

1000ug/mL IM Medium

MM salts

40mM MES milli-molar

10 milli-molar glucose - 80mg

0.5% w/v glycerol - 580ul

Vanillin 7.6mg/100ml

3.5% agar (1.5% does NOT SOLIDIFY)

IM Agar 

Small plates hold ~6.5mL

50mL each type

Liquid media:

IM Broth - 100mL

0.5% w/v glycerol 580uL

10 milli-molar dextrose 80mg

Vanillin:

Not soluble at 1000x!!*** - Make 100x

Method

Streaked out agrobacterium onto plates the day before on rif/spec agar.

Grew out in liquid culture: .62 on spec

Centrifuged 50mL and resuspended pellet in 100mL liquid culture medias (IM from paper, IM from ODIN)

Grow out mycelium on cellulose strips. Place strips into agrobacterium liquid culture for 4 hours, then plate on co-culture media.

After 5 days, move strips to selection plates containing hygromycin.

References: 

 https://www.sciencedirect.com/science/article/pii/S0944501310000571 

 https://academic.oup.com/femsle/article/362/5/fnv010/468761 

https://sci-hub.ru/10.1016/j.funbio.2020.08.001

 https://sci-hub.ru/https://link.springer.com/article/10.1007/s00294-005-0578-0 

 https://link.springer.com/article/10.1186/s40694-017-0035-0 

Feb. 20th

Moved filter paper to selection media.

Feb. 21st

 

Example of samples growing out on co-culture plates.