Part:BBa_K863101:Design

Cellulose binding Domain of Cellulomonas Fimi Exoglucanse (Freiburg-Standard)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal AgeI site found at 337
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The sequence starts with the rest of the Freiburg-prefix (ATG and NgoMIV-restriction-site). The A from the startcodon ATG is the last base of the XbaI restriction-site of the Standard 25 Prefix (Read here). I isolated 12 conserved Bases (4 AS) upstream of the cellulose binding domain and 9 bases downstream of the exoglucanase gene and added a short N-terminal (Glycine, Serine) linker. The sequence ends with the AgeI-restriction-site of the Freiburg-Suffix, to make in frame assembly possible.

Primers that were used to make this BioBrick:

CBDcex_Freiburg-Prefix	GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTCCGGCCGGGTGCCAGGTG
CBDcex_2AS-Link_Frei	CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCGCCGACCGTGCAGGGCGTGC

Source

Cloning-Vector with a CBDcex-Barnase fusion-protein

Contribution (Waterloo iGEM 2020)

McLean, B.W., Bray, M.R., Boraston, A.B., Gilkes, N.R., Haynes, C.A., & Kilburn, D.G. (2000). Analysis of binding of the family 2a carbohydrate -binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues. Protein Engineering, 13(11). 801-809.

Rodriguez, B., Kavoosi, M., Koska, J., Creagh, A.L., Kilburn, D.G., & Haynes, C.A. (2004). Inexpensive and Generic Affinity Purification of Recombinant Proteins Using a Family 2a CBM Fusion Tag. Biotechnology, 20. 1479 - 1489.