Part:BBa_K4883004
Ptef1-RIB1-PTV-ADE4-Tcyc1
This is a complete expression cassette, constructed by inserting RIB1-PTV-ADE4 (BBa_K4883003) between a strong promoter Ptef1 and a CYC1 terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3298
Illegal BamHI site found at 836 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 856
- 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. Co-overexpression of RIB1 (BBa_K4883001) and 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 co-overexpressing RIB1 and 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 co-overexpression of RIB1 and ADE4 increased vitamin B2 production by 131% 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 co-overexpression of RIB1 and ADE4 increased vitamin B2 production by 67% in steamed buns (p < 0.01).
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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