Difference between revisions of "Part:BBa K3866010"
(→‎Experimental Use and Experince)
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===Experimental Use and Experince===
 
===Experimental Use and Experince===
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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.
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After our first failed tyrosine assays measuring melanin at 400nm we conducted an SDS gel to examine wheather our protein is produced.
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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
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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>]]
 
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<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--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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*L: Lysate, I: Insoluble, S: Soluble
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*Ctr: BL21 DE3 with empty vector (a1R)
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*Tyr: BL21 DE3 with the Tyrosinase construct
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*1%, 0%: 1% and 0% concentration of L-arabinose, respectively
  
  
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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>]]
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[[File:T--Thessaly--SDStyr1.png|600px|thumb|none|<i><b>Fig.3:</b> This SDS page shows the tyrosinase production after induction with 1% L-arabinose </i>]]
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[[File:T--Thessaly--SDStyr2.png|600px|thumb|none|<i><b>Fig.4:</b> This SDS page shows that tyrosinase production is hindered after induction with 12% L-arabinose </i>]]
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<b>Conclusion after SDS page</b>
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<p>The enzyme is produced after induction with 1%  L-arabinose. 12% L-arabinose is really high concetration and propably toxic for the bacteria.
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The Tyrosinase assay was changed measuring at 670nm the melanin production.
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After 4 reapeats of the new Tyrosinase assay  we had a positive result shown bellow. This result is not reproducible.
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The experiments were conducted with 3 biological and with 3 technical replicates of each biological. Bellow Error bars are missing because of the ratio.
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[[File:T--Thessaly--tyrratio.png|600px|thumb|none|<i><b>Fig.5:</b> The ratio 670nm/600nm melanin production/cell growth of bacteria with Tyrosinase assay Buffer </i>]]
  
 
===Sequence and Features===
 
===Sequence and Features===

Revision as of 10:03, 4 October 2021


ParaBAD-tyrosinase:AIDA-terminator

Tyrosinase fused to AIDA autotranporterBBa_K3505006 for outer membrane exxpression[2]. Uder control of arabinose inducible promoterBBa_K3866000.

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 3 and 4 Expected bands (cut with EcoRV): 4835, 1956.

Experimental Use and Experince

After our first failed tyrosine assays measuring melanin at 400nm we conducted an SDS gel to examine wheather our protein is produced.


  • L: Lysate, I: Insoluble, S: Soluble
  • Ctr: BL21 DE3 with empty vector (a1R)
  • Tyr: BL21 DE3 with the Tyrosinase construct
  • 1%, 0%: 1% and 0% concentration of L-arabinose, respectively


Fig.3: This SDS page shows the tyrosinase production after induction with 1% L-arabinose


Fig.4: This SDS page shows that tyrosinase production is hindered after induction with 12% L-arabinose

Conclusion after SDS page

The enzyme is produced after induction with 1% L-arabinose. 12% L-arabinose is really high concetration and propably toxic for the bacteria. The Tyrosinase assay was changed measuring at 670nm the melanin production. After 4 reapeats of the new Tyrosinase assay we had a positive result shown bellow. This result is not reproducible. The experiments were conducted with 3 biological and with 3 technical replicates of each biological. Bellow Error bars are missing because of the ratio.

Fig.5: The ratio 670nm/600nm melanin production/cell growth of 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.