Difference between revisions of "Part:BBa K4883011"

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===Usage and Biology===
 
===Usage and Biology===
  
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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.   
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To mitigate vitamin B2 deficiency, we planned to make vitamin B2-enriched bread with engineered yeast, Saccharomyces cerevisiae. Overexpression of 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.   
  
  
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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).
 
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).
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<html>
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<body>
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<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/4883/wiki/bba-k4883011-1.png" style = "width:480px"></center>
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</div>
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<figcaption><center>Figure 1. Vitamin B2 contents in liquid YPD media containing engineered S. cerevisiae strains for 72h.  </center></figcaption>
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</figure>
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</body>
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</html>
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Results showed that overexpression of ADE4 increased vitamin B2 production by 44% after 72h in YPD.
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'''Vitamin B2 Production Tests in Steamed Buns'''
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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.
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<html>
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<body>
 +
<figure>
 +
<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/4883/wiki/bba-k4883011-2.png" style = "width:480px"></center>
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</div>
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<figcaption><center>Figure 2. Vitamin B2 contents in steamed buns leavened by engineered S. cerevisiae strains.  </center></figcaption>
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</figure>
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</body>
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</html>
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Results showed that overexpression of ADE4 increased vitamin B2 production by 19% in steamed buns (p > 0.05).
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==References==
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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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Revision as of 02:08, 6 October 2023


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2197
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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

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).

Figure 1. Vitamin B2 contents in liquid YPD media containing engineered S. cerevisiae strains for 72h.

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.

Figure 2. Vitamin B2 contents in steamed buns leavened by engineered S. cerevisiae strains.

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.