Difference between revisions of "Part:BBa K416000"

 
 
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<partinfo>BBa_K416000 short</partinfo>
 
<partinfo>BBa_K416000 short</partinfo>
  
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The Aga2 protein is fused in frame with a protein that the user wants to display on the yeast cellular surface. Yeast cells constitutively expressing Aga1 (strain EBY100) secrete the Aga2 fusion protein, which associates with Aga1 through two disulfide bonds and quaternary structure to anchor Aga2 to the surface. The fused protein then is displayed on the surface of the yeast cell.  
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In order to extracellularly display proteins on the yeast cell surface, the protein is fused to this Aga2 protein via a flexible linker. Once secreted, the Aga2 protein binds to the Aga1 protein on the yeast surface, anchoring the fusion onto the cell surface. Because the Aga2 protein must bind Aga1 to display, the chassis yeast strain must also express Aga1.
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The STOP codon at the end of the gene was removed from this CDS to allow for both N- and C-terminus fusions.
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<!-- Add more about the biology of this part here -->
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===Usage by GreatBay_United 2021===
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In 2021, GreatBay_United has used this part to display their protein complex onto the yeast surface. <br>
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The protein expression cassette was showed below:
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[[File:T--Greatbay United--p004.png|600px|thumb|center|the cassette of yeast surface display of GFP-HCV protease cut site-antiGFP complex]]
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The expression cassette was successfully transformed into yeast, and the relevant proteins were successfully expressed, see the  fluorescence image below:
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[[File:T--Greatbay United--p002.png|600px|thumb|center|GFP expression image]]
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In another cassette, GFP antibody (anti-GFP) were also expressed, if both complex and antiGFP were successfully displayed on the surface, they will be able to sense the HCV NS3 protease in solution. The protease would cut and release the antiGFP linked to GFP, and the solely expressed anti-GFP would had chance to connect with GFP. this interaction would lead to the macroscopical agglutination phenomenon when we mix two kinds of engineered yeast together and also HCV protease. The final agglutination could also prove that the signal peptide is working well.
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[[File:T--Greatbay United--p003.png|600px|thumb|center|anti-GFP expression cassette]]
  
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The STOP codon at the end of the CDS was removed to allow for N- and C-terminus fusions.  
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[[File:T--Greatbay United--p005.png|200px|thumb|center|the two types of yeast co-culture with HCV protease in the medium]]
  
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<!-- Add more about the biology of this part here
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[[File:T--Greatbay United--p006.png|200px|thumb|center|the two types of yeast co-culture without HCV protease in the medium]]
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===Usage and Biology===
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Latest revision as of 17:10, 20 October 2021

Aga2 CDS Responsible for Yeast Surface Display

In order to extracellularly display proteins on the yeast cell surface, the protein is fused to this Aga2 protein via a flexible linker. Once secreted, the Aga2 protein binds to the Aga1 protein on the yeast surface, anchoring the fusion onto the cell surface. Because the Aga2 protein must bind Aga1 to display, the chassis yeast strain must also express Aga1.

The STOP codon at the end of the gene was removed from this CDS to allow for both N- and C-terminus fusions.

Usage by GreatBay_United 2021

In 2021, GreatBay_United has used this part to display their protein complex onto the yeast surface.

The protein expression cassette was showed below:

the cassette of yeast surface display of GFP-HCV protease cut site-antiGFP complex

The expression cassette was successfully transformed into yeast, and the relevant proteins were successfully expressed, see the fluorescence image below:

GFP expression image

In another cassette, GFP antibody (anti-GFP) were also expressed, if both complex and antiGFP were successfully displayed on the surface, they will be able to sense the HCV NS3 protease in solution. The protease would cut and release the antiGFP linked to GFP, and the solely expressed anti-GFP would had chance to connect with GFP. this interaction would lead to the macroscopical agglutination phenomenon when we mix two kinds of engineered yeast together and also HCV protease. The final agglutination could also prove that the signal peptide is working well.

anti-GFP expression cassette
the two types of yeast co-culture with HCV protease in the medium
the two types of yeast co-culture without HCV protease in the medium

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]