Part:BBa_K4343118:Sequence, Features, and Subparts
PEX10 down-V2
URA gene is a commonly used screening marker in Y. lipolytica. Only strains with URA3 gene can grow in the medium without uracil. Since the Y. lipolytica strains we used were nutrition-deficient (uracil and leucine double deficient) and all plasmids we constructed (except BBa_K4343069) contained URA3gene, it could be verified whether the genome have been successfully edited. HisG tag were utilized in combination with URA3 gene to recycle this screening marker for further use in the same strain. To be more specific, 5-fluoroorotic acid (5-FOA) could be converted into a toxic form (e.g., 5-fluorouracil) by functional OMP decarboxylase encoded by URA3 gene, thus making yeast cells unable to grow. To survive the toxicity, yeast would miss the URA3 gene by HigG tag through negative screening. In this circumstance, URA deficient strain allows for the next transformation.