Composite

Part:BBa_K3505035

Designed by: Venetios Michelioudakis   Group: iGEM20_Thessaly   (2020-10-20)


pAndersonJ23115:RBS-tyrosinase:AIDA-terminator

Tyrosinase fused to AIDA autotranporterBBa_K3505006 for outer membrane exxpression[2]. Uder control of constitutive Anderson promoterBBa_K3505012.

Fig.1:The Tyrosine Tyrosinase Reporter Module


Usage and Biology

This Transcripion Unit is an electrochemical reporter module [1]. L-Tyrosine is converted to L-Dopa and L-Dopa quinone which is electrochemical detectable. This electochemical signal has the advantage of high quality and easy digitalization. The only advamtage is that is not well characterized.

Verification of the cloning

Fig.2:pAndersonJ23115:RBS-tyrosinase:AIDA-terminator Digested with BamHI. Expected bands 2847, 1883, 520, 353


Experimental Use and Experince

The Tyrosinase assay from the literature [1] is the addition of 1 g/L of L-Tyrosine into liquid cultures and incubation for 6 hours at 30 celcius.

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates

Fig.3: The Tyrosinase asssay checking the activity of the Tyrosinase

The constitutive Promoter led to toxicity so we plan next year to add an inducble one

Fig.4: The Bacterial Growth after 6 hours incubation at 30 celcius Bacteria with Tyrosinase assay Buffer

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1502
    Illegal EcoRI site found at 2198
    Illegal XbaI site found at 1106
    Illegal PstI site found at 1550
    Illegal PstI site found at 2136
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1502
    Illegal EcoRI site found at 2198
    Illegal NheI site found at 11
    Illegal NheI site found at 34
    Illegal PstI site found at 1550
    Illegal PstI site found at 2136
    Illegal NotI site found at 1274
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1502
    Illegal EcoRI site found at 2198
    Illegal BglII site found at 534
    Illegal BamHI site found at 517
    Illegal BamHI site found at 2400
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1502
    Illegal EcoRI site found at 2198
    Illegal XbaI site found at 1106
    Illegal PstI site found at 1550
    Illegal PstI site found at 2136
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1502
    Illegal EcoRI site found at 2198
    Illegal XbaI site found at 1106
    Illegal PstI site found at 1550
    Illegal PstI site found at 2136
    Illegal AgeI site found at 596
  • 1000
    COMPATIBLE WITH RFC[1000]

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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