Part:BBa_K4883011
Ptef1-ADE4-Tcyc1
This is a complete expression cassette consisting of a strong promoter Ptef1, an ADE4 gene, and a CYC1 terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2197
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 176
Usage and Biology
To mitigate vitamin B2 deficiency, we planned to make vitamin B2-enriched bread with engineered yeast, Saccharomyces cerevisiae. Overexpression of ADE4 (BBa_K4883000) should lead to the overproduction of vitamin B2 in S. cerevisiae. Ptef1 (BBa_K2407300) has been proven one of the strongest promoters of S. cerevisiae, so we used it for overexpression (Partow et al., 2010). A CYC1 terminator was also included to form the complete expression cassette.
Characterization
2023 Hangzhou-SDG Team characterized this part with vitamin B2 production
Vitamin B2 Production Tests in Liquid YPD Media
The WT S. cerevisiae S288C and the strain overexpressing ADE4 were inoculated into YPD media, and cultured at 30 ℃, 180 rpm. The riboflavin concentrations in the supernatants were measured using high-performance liquid chromatography (HPLC).
Results showed that overexpression of ADE4 increased vitamin B2 production by 44% after 72h in YPD.
Vitamin B2 Production Tests in Steamed Buns
To determine whether our engineered yeasts can help to make vitamin-B2-enriched food, we made steamed buns using our yeasts in the lab. The riboflavin concentrations in the buns were measured using HPLC.
Results showed that overexpression of ADE4 increased vitamin B2 production by 19% in steamed buns (p > 0.05).
References
Partow, S.; Siewers, V.; Bjørn, S.; Nielsen, J.; Maury, J. Characterization of Different Promoters for Designing a New Expression Vector in Saccharomyces Cerevisiae. Yeast 2010, 27 (11), 955–964. DOI:10.1002/yea.1806.
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