Difference between revisions of "Part:BBa K5321014"

 
 
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===Sequence and Features===
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<partinfo>BBa_K5321014 SequenceAndFeatures</partinfo>
  
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__NOTOC__
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===Usage and Biology===
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<partinfo>BBa_K5321014 short</partinfo>
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Given the challenges associated with the expression of proteins incorporating unnatural amino acids, we are exploring natural bioaffinity systems that can be constructed using simply fused proteins. We have selected streptavidin-biotin interactions for this purpose. Streptavidin (SA) is a tetrameric protein derived from the bacterium ''Streptomyces Avidini'', which exhibits extraordinary affinity for biotin. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained.
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Given the challenges associated with the expression of proteins incorporating unnatural amino acids, we are exploring natural bioaffinity systems that can be constructed using simply fused proteins. We have selected streptavidin-biotin interactions for this purpose. Streptavidin (SA) is a tetrameric protein derived from the bacterium ''Streptomyces Avidini'', which exhibits extraordinary affinity for biotin. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained. This approach simplifies the process while maintaining effective binding and functionality (Fig.1).
  
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===Usage and Biology===
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'''Figure 1 | Concept diagram of crosslinking between an aptamer and split protease via streptavidin-biotin interactions.''' The split protease is fused with streptavidin (SA), while the aptamer is biotinylated, allowing for a strong and specific binding that enhances protease functionality.<br>
  
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===Characterization===
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<span class='h3bb'>Sequence and Features</span>
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====Sequencing Verification====
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<partinfo>BBa_K5321014 SequenceAndFeatures</partinfo>
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After the Gibson assembly described above, sequencing was performed to verify the plasmids. The sequencing results confirmed that the construction was successful.
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'''Figure 2 | Sequencing verification of plasmids.''' (A-B) Sequencing results for the pET28a_SA_cPPVp plasmid, showing the N-terminal and C-terminal regions of streptavidin gene, respectively. (C-D) Sequencing results for the pET28a_SA_nPPVp plasmid, showing the N-terminal and C-terminal regions of streptavidin gene, respectively.
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====Protein Expression====
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Next, we transferred the correct plasmid into the expression strain of *E.coli* BL21(DE3). When the culture reached an OD600 of approximately 0.3 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C. However, the SDS-PAGE results showed no significant protein expression.
  
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'''Figure 3 | SDS analysis of the protein expression level and solubility for SA_cPPVp and SA_nPPVp.''' Lane1-3, total protein, supernatant after sonication, inclusion bodies after washing of SA_cPPVp, respectively. Lane4-6, total protein, supernatant after sonication, inclusion bodies after washing of SA_nPPVp, respectively.
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===Functional Parameters===
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<partinfo>BBa_K5321014 parameters</partinfo>
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Latest revision as of 13:52, 22 September 2024

SA_nPPVp

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 853
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 853
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 853
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 853
    Illegal NgoMIV site found at 508
    Illegal AgeI site found at 190
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Given the challenges associated with the expression of proteins incorporating unnatural amino acids, we are exploring natural bioaffinity systems that can be constructed using simply fused proteins. We have selected streptavidin-biotin interactions for this purpose. Streptavidin (SA) is a tetrameric protein derived from the bacterium Streptomyces Avidini, which exhibits extraordinary affinity for biotin. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained. This approach simplifies the process while maintaining effective binding and functionality (Fig.1).


Figure 1 | Concept diagram of crosslinking between an aptamer and split protease via streptavidin-biotin interactions. The split protease is fused with streptavidin (SA), while the aptamer is biotinylated, allowing for a strong and specific binding that enhances protease functionality.

Characterization

Sequencing Verification

After the Gibson assembly described above, sequencing was performed to verify the plasmids. The sequencing results confirmed that the construction was successful.

Figure 2 | Sequencing verification of plasmids. (A-B) Sequencing results for the pET28a_SA_cPPVp plasmid, showing the N-terminal and C-terminal regions of streptavidin gene, respectively. (C-D) Sequencing results for the pET28a_SA_nPPVp plasmid, showing the N-terminal and C-terminal regions of streptavidin gene, respectively.

Protein Expression

Next, we transferred the correct plasmid into the expression strain of *E.coli* BL21(DE3). When the culture reached an OD600 of approximately 0.3 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C. However, the SDS-PAGE results showed no significant protein expression.

Figure 3 | SDS analysis of the protein expression level and solubility for SA_cPPVp and SA_nPPVp. Lane1-3, total protein, supernatant after sonication, inclusion bodies after washing of SA_cPPVp, respectively. Lane4-6, total protein, supernatant after sonication, inclusion bodies after washing of SA_nPPVp, respectively.