Difference between revisions of "Part:BBa K3515015:Design"
 
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===Design Notes===
 
===Design Notes===
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Design Considerations: Added a Cysteine for immobilization (L664C) opposite to the active site region. Removed C223A, C316A, C370N, C673L, C96S, and C970S. to ensure only one cysteine would bind to an immobilization linker arm. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. All disulfides in Alpha-Klotho were kept to ensure proper structural folding. No modifications were made to the FRET acceptor or donor.
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Design Considerations: Added a Cysteine for immobilization (L664C) opposite to the active site region. Removed C223A, C316A, C370N, C673L, C963S, and C970S. to ensure only one cysteine would bind to an immobilization linker arm. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. All disulfides in Alpha-Klotho were kept to ensure proper structural folding. No modifications were made to the FRET acceptor or donor.
  
 
An mNeonGreen and mCherry fluorophore pair was chosen due to a variety of reasons, including but not limited to; stability at varying pH, brightness, and a large dynamic linear range due to its intensity.
 
An mNeonGreen and mCherry fluorophore pair was chosen due to a variety of reasons, including but not limited to; stability at varying pH, brightness, and a large dynamic linear range due to its intensity.

Latest revision as of 18:12, 29 June 2020


Alpha-Klotho Binding Protein with cysteine modification(s) and FRET to monitor phosphate levels usin


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2337
    Illegal XhoI site found at 1876
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 871
    Illegal AgeI site found at 1698
    Illegal AgeI site found at 2567
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Design Considerations: Added a Cysteine for immobilization (L664C) opposite to the active site region. Removed C223A, C316A, C370N, C673L, C963S, and C970S. to ensure only one cysteine would bind to an immobilization linker arm. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. All disulfides in Alpha-Klotho were kept to ensure proper structural folding. No modifications were made to the FRET acceptor or donor.

An mNeonGreen and mCherry fluorophore pair was chosen due to a variety of reasons, including but not limited to; stability at varying pH, brightness, and a large dynamic linear range due to its intensity.

Spectragreencherry.png

Fluorophore spectrum of the mNeonGreen and mCherry fluorophores. Optical density data for wavelengths 300 nm to 750 nm were plotted for mNeonGreen and mCherry fluorescent proteins, in green and red colors, respectively. Data was obtained from www.fpbase.org. Excitation and emission peaks are labelled as EX and EM, respectively, for each fluorescent protein. Triangular dashed region shows the approximate fluorophore pair overlap, indicating that at an appropriate distance, energy transfer will occur between the donor (mNeonGreen) and acceptor (mCherry).

Source

The source of this part is its sequence retrieved from the European Nucleotide Archive (M37722.1) along with our own modifications.

References

Chen, G., Liu, Y., Goetz, R., Fu, L., Jayaraman, S., Hu, M.C., Moe, O.W., Liang, G., Li, X. and Mohammadi, M., 2018. α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling. Nature, 553(7689), pp.461-466.