Reporter

Part:BBa_K3505006

Designed by: Venetios Michelioudakis   Group: iGEM20_Thessaly   (2020-10-11)
Revision as of 10:31, 13 October 2020 by Venetios (Talk | contribs)


Tyr1-AIDAc Tyrosinase fused to membrane protein AIDAc. GB compatible B3-B5

Tyrosinase catalyzes the conversion of L-Tyrosine to L-Dopa and L-Dopa quinone. These derivatives can be detected electochemically, producing peak currents.[1]

Usage and Biology

AIDA is native E.coli outer membrane protein. AIDA's CDS constists of 3 parts: [2]

  • A Signal Peptide that is cleaved in order the rest of the protein to be transported to the outer membrane.
  • The AIDAc is the autotransporter with the transmembrane part.
  • The passeger of AIDA which is the part that is on the exterior side.

We placed as passenger the Tyr1 gene coding to Rhizobium etli tyrosinase Because is the smallest of all tyrosinases (34kDa) and lack cysteines[3] compared to the melA tyrosinase BBa_K193600 used in iGEM before.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in pUPD2 as Level 0 and has overhangs compatible for Golden Braid cloning. The CDS has position B3-B5.

T--Thessaly--GB-AATG-GCTT.jpeg

Figure 1. The GB2.0 grammar

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1439
    Illegal EcoRI site found at 2135
    Illegal XbaI site found at 1043
    Illegal PstI site found at 1487
    Illegal PstI site found at 2073
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1439
    Illegal EcoRI site found at 2135
    Illegal PstI site found at 1487
    Illegal PstI site found at 2073
    Illegal NotI site found at 1211
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1439
    Illegal EcoRI site found at 2135
    Illegal BglII site found at 471
    Illegal BamHI site found at 454
    Illegal BamHI site found at 2337
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1439
    Illegal EcoRI site found at 2135
    Illegal XbaI site found at 1043
    Illegal PstI site found at 1487
    Illegal PstI site found at 2073
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1439
    Illegal EcoRI site found at 2135
    Illegal XbaI site found at 1043
    Illegal PstI site found at 1487
    Illegal PstI site found at 2073
    Illegal AgeI site found at 533
  • 1000
    COMPATIBLE WITH RFC[1000]

Source

  • Tyr1 from Rhizobium etli in the supplementary of [3]
  • AIDA from E.coli in the supplementary of [2]

References

  • [1] Eric VanArsdale, David Hörnström, Gustav Sjöberg, Ida Järbur, Juliana Pitzer, Gregory F. Payne, Antonius J. A. van Maris, and William E. Bentley. A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks.ACS Synthetic Biology 2020 9 (5), 1117-1128

DOI: 10.1021/acssynbio.9b00469

  • [2] Gustavsson, M., Hörnström, D., Lundh, S. et al. Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior. Sci Rep 6, 36117 (2016). https://doi.org/10.1038/srep36117
  • [3] David Hörnström, Gen Larsson, Antonius J.A. van Maris, Martin Gustavsson, Molecular optimization of autotransporter-based tyrosinase surface display, Biochimica et Biophysica Acta (BBA) - Biomembranes , Volume 1861, Issue 2, 2019, Pages 486-494, https://doi.org/10.1016/j.bbamem.2018.11.012.

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