In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.
In this presentation, we illustrated how to use the budding yeast Saccharomyces cerevisiae as a heterologous system for the expression of type III bacterial effector proteins and how to identify effector-induced growth inhibition phenotypes. Importantly, these phenotypes can be utilized in genetic screens to identify suppressors of the negative impact of effectors on yeast growth. Suppressors may represent either direct targets of the effector studied or proteins that participate in cellular processes affected b…
This work was supported by the Israel Science Foundation.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Yeast extract | Difco | 212750 | ||
Peptone | Difco | 211677 | ||
D-glucose | Sigma | G5767 | ||
Agar | Difco | 214010 | ||
Sodium hydroxide (NaOH) | Sigma | S8045 | ||
Yeast nitrogen base w/o amino acids | Difco | 291940 | ||
Yeast synthetic drop-out medium supplement | Sigma | Y2001 | ||
D-galactose | Sigma | G0750 | >99%; <0.1% glucose | |
D-raffinose | Sigma | R0250 | >98% | |
L-leucine | Sigma | L8000 | ||
Uracil | Sigma | U0750 | ||
L-tryptophan | Sigma | T0254 | ||
L-histidine | Sigma | H6034 | ||
DNA, single stranded, from salmon testes | Sigma | D7656 | ||
Dimethyl sulfoxide (DMSO) | Sigma | D5879 | Desiccate | |
Hydrochloric acid (HCl) | Sigma | H1758 | ||
Polyethylene glycol (PEG) 3350 | Sigma | P4338 | ||
Lithium acetate (LiAc) | Sigma | L4958 | ||
Tris (base) | J.T. Baker | 4109-02 | ||
Ethylenediamine-tetraacetic acid (EDTA) | Sigma | E5134 | ||
β-mercaptoethanol | Sigma | M6250 | ||
Glycerol | Sigma | G5516 | ||
Bromophenol blue | Sigma | B6131 | ||
Dodecyl sulfate sodium salt (SDS) | Merck | 8.22050.1000 | ||
Centrifuge tubes (15 ml) | Corning | 430052 | Sterile | |
Spectrophotometer cuvette (10x4x45 mm) | Sarstedt | 67.742 | ||
Inoculation loop | Sigma | Z643009 | Sterile | |
Parafilm | Sigma | P7543 | ||
pH indicator strip, pH 6.5-10.0 | Merck | 1.09543.0001 |