Part:BBa_K3505035
pAndersonJ23115:RBS-tyrosinase:AIDA-terminator
Tyrosinase fused to AIDA autotranporterBBa_K3505006 for outer membrane exxpression[2]. Uder control of constitutive Anderson promoterBBa_K3505012.
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
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
The constitutive Promoter led to toxicity so we plan next year to add an inducble one
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 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.
None |