Part:BBa_K4229074

BMC-T1_Y96TAG

BMC-T1-Y96TAG Short description: The Y96TAG 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_Y96TAG can only be completely synthesized upon successful amber stop codon suppression. Hence, BMC-T1_Y96TAG 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_Y96TAG 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_Y96TAG 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.

The plasmid containing this protein was kindly sent to us by the Kerfeld group.

H. Kirst and C. A. Kerfeld, “Bacterial microcompartments: Catalysis-enhancing metabolic modules for next-generation metabolic and biomedical engineering,” BMC Biol., vol. 17, no. 1, pp. 1–11, 2019, DOI: 10.1186/s12915-019-0691-z.

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 36
  Illegal AgeI site found at 510
  Illegal AgeI site found at 618
  
• 1000
  COMPATIBLE WITH RFC[1000]