Coding

Part:BBa_K4229071

Designed by: Nikita Edel   Group: iGEM22_Freiburg   (2022-10-04)


BMC-T1_F8TAG



BMC-T1-F8TAG Short description: The F8TAG variant of BMC-T1(BBa_K4229037) was created using site-directed mutagenesis, aiming to insert a non-canonical amino acid at position 8 of the peptide sequence by amber stop codon suppression. The BMC-T1_F8TAG can only be completely synthesized upon successful amber stop codon suppression. Hence, BMC-T1_F8TAG can be used to create a ncAA modified shell protein for the assembly of a synthetic carboxysome.


Usage: Besides the well-known 20 canonical amino acids, a variety of non-canonical amino acids can be used to modify proteins. One way of incorporating non-canonical amino acids is via the amber stop codon suppression technology. BMC-T1_F8TAG is a mutated version of the BMC-T1 protein, necessary to form the synthetic BMC of the myxobacterium Haliangium ochraceum. Without successful incorporation, a non-functional version of BMC-T1 will be translated, and the synthetic carboxysome cannot form. However, after successful incorporation of the non-canonical amino acid, translation will go on as normal and the synthetic carboxysome can form.


Characterization: The incorporation of non-canonical amino acids into BMC-T1_ F8TAG was characterized using SDS-PAGE, Western blot, and antibody decoration. As an orthogonal translation system, an additional aminoacyl-tRNA synthetase and its corresponding tRNA are required, which in this case correspond to the pAzF tRNA synthetase (pAzFRS), which incorporates 4-azido-l-phenylalanine.

Figure text: SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8, 35, 78 and 96 of the BMC-T1 protein. The cells grew until OD 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used for orientation. To purify the protein, the His-Spin Protein Miniprep from Zymo Research was used.

























Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 706
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 33
    Illegal AgeI site found at 510
    Illegal AgeI site found at 618
  • 1000
    COMPATIBLE WITH RFC[1000]


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