Integrating design-of-experiments (DOE) optimization and risk assessment towards a safe and simplified electroporation protocol for Toxoplasma gondii.

Genetic manipulation of Toxoplasma gondii presents unique challenges due to its obligatory intracellular nature and relatively rapid growth. Electroporation is the main technique used to introduce genetic modifications into T. gondii. However, the existing protocols require an electroporation buffer comprised of eight components and involving multiple steps of preparation. Optimizing electroporation protocols, including a readily available buffer is crucial for achieving efficient transfection while simplifying the overall process. In this study, we present a modified Opti-MEM I based electroporation buffer that matches cytomix in performance with significantly reduced variability. We also develop a novel scoring method (etScore) to reproducibly quantify electroporation performance, combining transgene gene expression with cell viability. We also couple the experimental work with a corresponding systematic risk assessment and argue for routine use of such tools in similar contexts. We anticipate this protocol will make genetic modification of T. gondii more accessible to the international community, accelerating drug and vaccine research.