SheinerLab
One-Step strategy to generate conditional mutants in Toxoplasma gondii
This system is based on two important technical innovations:
1. The establishment of a Toxoplasma line expressing a tetracyclin regulatable trans-activator
(Meissner et al; Science. 2002 Oct 25;298(5594):837-40)
2. The finding that null Ku80 Toxoplasma mutants are viable and are more susceptible to homologous recombination
(Huynh et al; Eukaryot Cell. 2009 Apr;8(4):530-9; Fox et al; Eukaryot Cell. 2009 Apr;8(4):520-9)
Dr Sheiner engineered a line that contain both these genetic manipulations. This line allows a single step replacment of the endogenous promoter of a gene of interest (GOI) with an inducible promoter with high efficiency.
This work was published here:
Sheiner et al; PLoS Pathog. 2011 Dec;7(12):e1002392
Download promoter replacement plasmid map here:
Donwload UPRT directed complementation plasmid here:
UPDATE:
Nowadays we generate promoter replacement using CRISPR/Cas9 to enhance the integration of a PCR product that carries the regulatable promoter and a selectable marker flanked with 50bp of homology to the endogenous promoter region.
If you are interested in any of the reagents don't hesitate to contact the Sheiner lab: Lilach.Sheiner@glasgow.ac.uk
Inducible over-expression in Toxoplasma gondii
We generated an inducible "turn-on" system based on (Andenmatten et al; Nat Methods. 2013 Feb;10(2):125-7. doi: 10.1038/nmeth.2301):
Fluorescent redox-sensor for Toxoplasma gondii
We generated a live redox sensor in Toxoplasma. This is based on the same roGFP molecules used in mammalian cell:
More details about roGFP and inducible over-expression here:
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006836
A way to report on mitochondrial translation defects in Toxoplasma gondii
GeneOff killerRed (red) expresses
GeneOn killerRed doesn't expresses, gene with myc tag (green) expresses
+ Rapamycine
The rational for this assay is that complex IV activity depends on mitochondrial translation for assembly, while complex V, which components are all nuclear encoded, doesn't - thus comparing the activity of both complexes provides a proxy to identify mitochondrial translation defects.