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Electroporation is the main technique used to introduce genetic modifications into\n                    <jats:italic>T. gondii<\/jats:italic>\n                    . 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\n                    <jats:italic>T. gondii<\/jats:italic>\n                    more accessible to the international community, accelerating drug and vaccine research.\n                  <\/jats:p>","DOI":"10.1371\/journal.pntd.0014194","type":"journal-article","created":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T17:52:49Z","timestamp":1775670769000},"page":"e0014194","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrating design-of-experiments (DOE) optimization and risk assessment towards a safe and simplified electroporation protocol for Toxoplasma gondii"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9273-675X","authenticated-orcid":true,"given":"Pratik 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