Part:BBa_K3866010

ParaBAD-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 disadvantage is that is not well characterized.

Verification of the cloning

Fig.2:paraBAD:RBS-tyrosinase:AIDA-terminator Positive clone 4 and 5 Expected bands (cut with EcoRV): 4835, 1956.

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

Fig.5: 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 1852
  Illegal EcoRI site found at 2548
  Illegal XbaI site found at 1456
  Illegal PstI site found at 1900
  Illegal PstI site found at 2486
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1852
  Illegal EcoRI site found at 2548
  Illegal PstI site found at 1900
  Illegal PstI site found at 2486
  Illegal NotI site found at 1624
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1852
  Illegal EcoRI site found at 2548
  Illegal BglII site found at 884
  Illegal BamHI site found at 867
  Illegal BamHI site found at 2750
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1852
  Illegal EcoRI site found at 2548
  Illegal XbaI site found at 1456
  Illegal PstI site found at 1900
  Illegal PstI site found at 2486
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1852
  Illegal EcoRI site found at 2548
  Illegal XbaI site found at 1456
  Illegal PstI site found at 1900
  Illegal PstI site found at 2486
  Illegal AgeI site found at 946
  
• 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.