Difference between revisions of "Part:BBa K863101:Design"

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'''Improvement (Waterloo iGEM 2020)'''
 
'''Improvement (Waterloo iGEM 2020)'''
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Courtade, G.; Forsberg, Z.; Heggset, E. B.; Eijsink, V. G. H. & Aachmann, F. L. (2018). The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation. ''The Journal of Biological Chemistry, 293'', 13006-13015.
 
Courtade, G.; Forsberg, Z.; Heggset, E. B.; Eijsink, V. G. H. & Aachmann, F. L. (2018). The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation. ''The Journal of Biological Chemistry, 293'', 13006-13015.

Revision as of 02:21, 21 October 2020

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

References

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.

Improvement (Waterloo iGEM 2020)

Courtade, G.; Forsberg, Z.; Heggset, E. B.; Eijsink, V. G. H. & Aachmann, F. L. (2018). The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation. The Journal of Biological Chemistry, 293, 13006-13015.