Coding
P0DTR4

Part:BBa_K4726000:Design

Designed by: Benjamin Ouellet   Group: iGEM23_ULaval   (2023-10-01)
Revision as of 01:25, 10 October 2023 by Benjamin Ouellet (Talk | contribs) (→‎Design Notes)

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N-acetyl-α-D-galactosamine deacetylase_Flavonifractor plautii


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
    Illegal NgoMIV site found at 1584
    Illegal NgoMIV site found at 1894
  • 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 Flavonifractor plautii, a bacterium from the human gut microbiota. It encodes a N-acetyl-α-D-galactosamine deacetylase.

The amino sequence is:

MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSD SAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDG VHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGG GVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFIT AIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSD DGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGR NPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPG SSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAEN SFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPV DTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIA VVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKD ELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVA SGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIE PAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVG TAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGK HTIRIVCVSPVVDFDYFSYVGEHHHHHH

References

[1] Rahfeld P, Sim L, Moon H, Constantinescu I, Morgan-Lang C, Hallam SJ, Kizhakkedathu JN, Withers SG. An enzymatic pathway in the human gut microbiome that converts A to universal O type blood. Nat Microbiol. 2019 Sep;4(9):1475-1485. doi: 10.1038/s41564-019-0469-7. Epub 2019 Jun 10. PMID: 31182795.