Part:BBa_K3866010
ParaBAD-tyrosinase:AIDA-terminator
Tyrosinase fused to AIDA autotranporterBBa_K3505006 for outer membrane exxpression[2]. Uder control of arabinose inducible promoterBBa_K3866000.
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
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%, 12% and 0% concentration of 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.[4]
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.
- Ctr: BL21 with empty vector (a1R)
- Tyr: BL21 with the Tyrosinase construct
- 1% and 0% concentration of L-arabinose, respectively
- -1: Tyrosinase assay buffer (contains tyrosine that serves as a substrate for melanin production)
- -2: Washing buffer (no tyrosine)
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.
- [4] della-Cioppa, G., Garger, S. J., Sverlow, G. G., Turpen, T. H., & Grill, L. K. (1990). Melanin production in Escherichia coli from a cloned tyrosinase gene. Bio/technology (Nature Publishing Company), 8(7), 634–638. https://doi.org/10.1038/nbt0790-634
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