Difference between revisions of "Part:BBa K4883006"

 
 
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<partinfo>BBa_K4883006 short</partinfo>
 
<partinfo>BBa_K4883006 short</partinfo>
  
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Insert a 2A self-cleaving peptide, ERBV-1, between RIB1 and RIB7. ERBV-1 helps to achieve bicistronic gene expression in Saccharomyces cerevisiae. Use a strong constitutive promoter Ptef1 to overexpress RIB1 and RIB7.
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This is a complete expression cassette, constructed by inserting RIB1-ERBV-1-RIB7 ([https://parts.igem.org/Part:BBa_K4883005 BBa_K4883005]) between a strong promoter Ptef1 and a CYC1 terminator.  
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K4883006 parameters</partinfo>
 
<partinfo>BBa_K4883006 parameters</partinfo>
 
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<!-- Add more about the biology of this part here-->
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===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. Co-overexpression of RIB1 ([https://parts.igem.org/Part:BBa_K4883001 BBa_K4883001]) and RIB7 ([https://parts.igem.org/Part:BBa_K4883002 BBa_K4883002]) 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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===Characterization===
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'''2023 Hangzhou-SDG Team characterized this part with vitamin B2 production'''
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'''Vitamin B2 Production Tests in Liquid YPD Media'''
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The WT S. cerevisiae S288C and the strain co-overexpressing RIB1 and RIB7 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-k4883006-1.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 co-overexpression of RIB1 and RIB7 increased vitamin B2 production by 96% 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>
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<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/4883/wiki/bba-k4883006-2.png" style = "width:280px"></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 co-overexpression of RIB1 and RIB7 increased vitamin B2 production by 25% 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.

Latest revision as of 02:59, 6 October 2023


Ptef1-RIB1-ERBV-1-RIB7-Tcyc1

This is a complete expression cassette, constructed by inserting RIB1-ERBV-1-RIB7 (BBa_K4883005) between a strong promoter Ptef1 and a CYC1 terminator.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1743
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2494
    Illegal BamHI site found at 836
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 856
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 176
    Illegal BsaI.rc site found at 1675



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 RIB7 (BBa_K4883002) 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 RIB7 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 co-overexpression of RIB1 and RIB7 increased vitamin B2 production by 96% 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 co-overexpression of RIB1 and RIB7 increased vitamin B2 production by 25% 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.