Difference between revisions of "Part:BBa K4883004"
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<!-- Add more about the biology of this part here --> | <!-- Add more about the biology of this part here --> | ||
===Usage and Biology=== | ===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 ([https://parts.igem.org/Part:BBa_K4883001 BBa_K4883001]) and ADE4 ([https://parts.igem.org/Part:BBa_K4883000 BBa_K4883000]) should lead to the overproduction of vitamin B2 in S. cerevisiae. Ptef1 ([https://parts.igem.org/Part:BBa_K2407300 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). | ||
+ | |||
+ | <html> | ||
+ | <body> | ||
+ | <figure> | ||
+ | <div class = "center"> | ||
+ | <center><img src = "https://static.igem.wiki/teams/4883/wiki/bba-k4883004-1.png" style = "width:480px"></center> | ||
+ | </div> | ||
+ | <figcaption><center>Figure 1. Vitamin B2 contents in liquid YPD media containing engineered S. cerevisiae strains for 72h. </center></figcaption> | ||
+ | </figure> | ||
+ | </body> | ||
+ | </html> | ||
+ | |||
+ | 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. | ||
+ | |||
+ | |||
+ | <html> | ||
+ | <body> | ||
+ | <figure> | ||
+ | <div class = "center"> | ||
+ | <center><img src = "https://static.igem.wiki/teams/4883/wiki/bba-k4883004-2.png" style = "width:480px"></center> | ||
+ | </div> | ||
+ | <figcaption><center>Figure 2. Vitamin B2 contents in steamed buns leavened by engineered S. cerevisiae strains. </center></figcaption> | ||
+ | </figure> | ||
+ | </body> | ||
+ | </html> | ||
+ | |||
+ | 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. |
Revision as of 02:54, 6 October 2023
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.