Hi, I am doing Agrobacterium-mediated transformation of V. inaequalis with the LBA4404 strain. However, I am encountering problems with LBA4404 aggregating/forming clumps when growing in induction media (see photo). Does anyone know what can be causing aggregation? I noticed that when this happens my fungal transformation fails.

The protocol that I am using is the following:

- One day before transformation (morning) inoculate Agro strain in YM media (25 ml in 250 ml flask). Grow at 28C, shacking 220 rpm. During this stage the Agro grows very well, does not form any clump and the culture usually reaches an OD600: 1- 1.8.

- Spin-down culture 4500 rpm, 10 minutes at 4C.

- Resuspend in 15 ml Induction media (IM).

- Repeat washing step.

- Measure OD600. Dilute culture to a final OD600 of 0.15.

- Grown x2 cultures (15ml in a 50 ml Eppendorf tube) with acetosynringone (30 uL of 0.1M stock, made fresh) and 15 ug/ml chloramphenicol. Grow at 28C, 22O rpm.

-Incubate Agro in IM with acetosyringone + antibiotic for 5 to 6 hours 28C, 220 rpm.

Here the problems start, at the 1st hour of incubation the Agro is already forming aggregates. I`ve also tried to incubate it in a 250 mL conical flask but does not help.

The induction media recipe that I use is:

Liquid induction media (Prepare fresh, afternoon before transformation).

· Minimal salts: g/L

FeSO4∙7H20 0.0025 g

CaCl2∙2H20 0.05 g

K2HPO4-3 H20 2.05 g

KH2PO4 1.45 g

NaCl 0.15 g

MgSO4∙7H2O 0.5 g

(NH4)2SO4 0.5 g

· MES-H20 7.8g (40mM)

· D-glucose 1.8g (10mM)

· Glycerol 5ml 0.5% w/v

Adjust pH 5.3 (with HCl). Filter sterilize.

Does anyone know what can be causing the Agro strain to aggregate? Thank you :)