Coding
GH110b

Part:BBa_K4726002:Design

Designed by: Benjamin Ouellet   Group: iGEM23_ULaval   (2023-10-01)
Revision as of 01:12, 10 October 2023 by ArnaudG (Talk | contribs) (→‎Source)

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α-galactosidase GH110b_Bacteroides fragilis


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1213
    Illegal EcoRI site found at 1714
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1213
    Illegal EcoRI site found at 1714
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1213
    Illegal EcoRI site found at 1714
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1213
    Illegal EcoRI site found at 1714
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1213
    Illegal EcoRI site found at 1714
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In order to clone this genetic construct onto our expression vector (pET-3a) we added NdeI site in 5' and BamHI site in 3' of the gene. We added TATATA sequences before the NdeI site and after the BamHI site to assist with the restriction digestion (those sites are not on the part sequence). The part sequence included a 6x His-tag on the C-terminus of the protein.

Source

This gene originates from Bacteroides fragilis, a bacterium from the human gut microbiota. It encodes an α-galactosidase.

The amino sequence is:

MKTILLFALSLLLSLSVSDVCAQERVYDISQFGLKANSKKNASPVVRKAI AKIKAECRDGEKVILRFPAGRYNFHEAGSTVREYYISNHDQDNPKKVGIA LEDMKNLTIDGQGSEFVFYGRMIPVSLLRSENCVLKNFSIDFEQPHIAQV QVVENDPEKGITFEPAPWVDYRISKDSVFEGLGEGWVMRYSWGIAFDGKT KHVVYNTSDIGCPTKGAFEVAPRRICSPKWKDARLVPGTVVAMRGWGRPT PGIFMSHDVNTSLLDVKVHYAEGMGLLAQLCEDITLDGFGVCLKGDNDPR YFTTQADATHFSGCKGKIVSKNGLYEGMMDDAINVHGTYLKVIKRVDDHT LIGRYMHDQSWGFEWGRPGDDVQFVRSETMELIGKQNQITAIRPYDKGEI RGAREFSITFKEAIDPAINEKSGFGIENLTWTPEVLFAGNTIRNNRARGT LFSTPKKTVVEDNLFDHTSGTAILLCGDCNGWFETGACRDVTIRRNRFIN ALTNMFQFTNAVISIYPEIPNLKDQQKYFHGGKDGGIVIEDNEFDTFDAP ILYAKSVDGLIFRNNVIKTNTEFKPFHWNKDRFLLERVTNVKISEHHHHH H

References

[1] Liu QP, Yuan H, Bennett EP, Levery SB, Nudelman E, Spence J, Pietz G, Saunders K, White T, Olsson ML, Henrissat B, Sulzenbacher G, Clausen H. Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen. J Biol Chem. 2008 Mar 28;283(13):8545-54. doi: 10.1074/jbc.M709020200. Epub 2008 Jan 28. PMID: 18227066; PMCID: PMC2417185.