Difference between revisions of "Part:BBa K3866010"
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Tyrosinase fused to AIDA autotranporter<bbpart>BBa_K3505006</bbpart> for outer membrane exxpression[2]. Uder control of constitutive Anderson promoter<bbpart>BBa_K3505012</bbpart>.
 
Tyrosinase fused to AIDA autotranporter<bbpart>BBa_K3505006</bbpart> for outer membrane exxpression[2]. Uder control of constitutive Anderson promoter<bbpart>BBa_K3505012</bbpart>.
  
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[[File:T--Thessaly--tyr.png|600px|thumb|none|<i><b>Fig.1:</b>The Tyrosine Tyrosinase Reporter Module </i>]]
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[[File:T--Thessaly--aractyr.png|600px|thumb|none|<i><b>Fig.1:</b>The Tyrosine Tyrosinase Reporter Module </i>]]
  
  
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===Verification of the cloning===
 
===Verification of the cloning===
  
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[[File:T--Thessaly--tyr11.png|600px|thumb|none|<i><b>Fig.2:</b>pAndersonJ23115:RBS-tyrosinase:AIDA-terminator Digested with BamHI. Expected bands 2847, 1883, 520, 353 </i>]]
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[[File:T--Thessaly--tyrgel.png|600px|thumb|none|<i><b>Fig.2:</b>paraBAD:RBS-tyrosinase:AIDA-terminator Positive clone 4 and 5 Expected bands (cut with EcoRV): 4835, 1956.</i>]]
  
  
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<p>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
 
<p>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
  
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[[File:T--Thessaly--ty.png|600px|thumb|none|<i><b>Fig.3:</b> The Tyrosinase asssay checking the activity of the Tyrosinase </i>]]
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[[File:T--Thessaly--SDStyr1.png|600px|thumb|none|<i><b>Fig.3:</b> The Tyrosinase asssay checking the activity of the Tyrosinase </i>]]
 
<b>The constitutive Promoter led to toxicity so we plan next year to add an inducble one </b>
 
<b>The constitutive Promoter led to toxicity so we plan next year to add an inducble one </b>
  
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[[File:T--Thessaly--tyg.png|600px|thumb|none|<i><b>Fig.4:</b> The Bacterial Growth after 6 hours incubation at 30 celcius  Bacteria with Tyrosinase assay Buffer </i>]]
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[[File:T--Thessaly--SDStyr2.png|600px|thumb|none|<i><b>Fig.4:</b> The Bacterial Growth after 6 hours incubation at 30 celcius  Bacteria with Tyrosinase assay Buffer </i>]]
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[[File:T--Thessaly--tyrratio.png|600px|thumb|none|<i><b>Fig.5:</b> The Bacterial Growth after 6 hours incubation at 30 celcius  Bacteria with Tyrosinase assay Buffer </i>]]
  
 
===Sequence and Features===
 
===Sequence and Features===

Revision as of 08:58, 4 October 2021


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 advamtage 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.