Difference between revisions of "Part:BBa K4883010"

 
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<partinfo>BBa_K4883010 short</partinfo>
 
<partinfo>BBa_K4883010 short</partinfo>
  
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Insert two 2A self-cleaving peptides, ERBV-1 and PTV, between RIB1, RIB7, and ADE4. ERBV-1 and PTV help to achieve bicistronic gene expression in Saccharomyces cerevisiae. Use a strong constitutive promoter Ptef1 to overexpress RIB1, RIB7, and ADE4.  
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This is a complete expression cassette, constructed by inserting RIB1-ERBV-1-RIB7-PTV-ADE4 ([https://parts.igem.org/Part:BBa_K4883009 BBa_K4883009]) between a strong promoter Ptef1 and a CYC1 terminator.  
  
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K4883010 parameters</partinfo>
 
<partinfo>BBa_K4883010 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]), RIB7 ([https://parts.igem.org/Part:BBa_K4883002 BBa_K4883002]), 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.
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===Characterization===
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‘’‘2023 Hangzhou-SDG Team characterized this part with vitamin B2 production’‘’
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To find the strain with the highest vitamin B2 production, we constructed different strains overexpressing different combinations of RIB1, RIB7, and ADE4 ([https://parts.igem.org/Part:BBa_K4883011 BBa_K4883011], [https://parts.igem.org/Part:BBa_K4883012 BBa_K4883012], [https://parts.igem.org/Part:BBa_K4883013 BBa_K4883013], [https://parts.igem.org/Part:BBa_K4883004 BBa_K4883004], [https://parts.igem.org/Part:BBa_K4883006 BBa_K4883006], [https://parts.igem.org/Part:BBa_K4883008 BBa_K4883008], [https://parts.igem.org/Part:BBa_K4883010 BBa_K4883010]).
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‘’‘Vitamin B2 Production Tests in Liquid YPD Media’‘’
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The WT S. cerevisiae S288C and the engineered strains 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).

Revision as of 03:10, 6 October 2023


Ptef1-RIB1-ERBV-1-RIB7-PTV-ADE4-Tcyc1

This is a complete expression cassette, constructed by inserting RIB1-ERBV-1-RIB7-PTV-ADE4 (BBa_K4883009) 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 4090
    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), RIB7 (BBa_K4883002), 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’‘’

To find the strain with the highest vitamin B2 production, we constructed different strains overexpressing different combinations of RIB1, RIB7, and ADE4 (BBa_K4883011, BBa_K4883012, BBa_K4883013, BBa_K4883004, BBa_K4883006, BBa_K4883008, BBa_K4883010).

‘’‘Vitamin B2 Production Tests in Liquid YPD Media’‘’

The WT S. cerevisiae S288C and the engineered strains 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).