Part:BBa_K3843002

1UTM-HRP (fusion)

1UTM-HRP is a translational fusion of Atlantic salmon trypsin (1UTM) and horseradish peroxidase (HRP). 1UTM can bind to PEA (a natural inhibitor of salmon trypsin), and HRP is a chemiluminescent enzyme. A 6-His tag followed by a TEV protease linker was added at the N-terminus to allow for purification by Ni-NTA affinity chromatography. At the C-terminus, an Avi-tag was appended, allowing for biotinylation. A flexible, moderately long (13 AA), and polar linker (consisting of Ser and Thr) separated 1UTM and HRP, allowing for proper functioning and stability of the fusion protein in aqueous solution. The resulting fusion protein can be used for the detection of PEA in a diagnostic microfluidic assay that produces a quantifiable chemiluminescent signal, which was the basis of Waterloo iGEM 2021's project.

The following image depicts the 1UTM-HRP fusion (with 1UTM on the left and HRP on the right). The 3D model was obtained by multi-domain structural modelling on UCSF Chimera (Pettersen et al., 2004).

References

Cull, M. G., & Schatz, P. J. (2000). Biotinylation of proteins in vivo and in vitro using Small Peptide tags. Methods in Enzymology, (326), 430–440. https://doi.org/10.1016/s0076-6879(00)26068-0

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C. & Ferrin, T. E. UCSF Chimera--a visualization system for exploratory research and analysis (Version 1.15). J Comput Chem. 2004; 25(13): 1605-1612. https://www.ncbi.nlm.nih.gov/pubmed/15264254

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1113
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1077
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1249