Part:BBa_K4165177

Biobrick His - Trim

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), RBS 3 (BBa_K4165263), 6x His-tag (BBa_K4165020), Trim21 improved (BBa_K4165001), and T7 terminator (BBa_K731721), The His tag was attached to the Trim21 (improved) coding sequence to serve in the purification using NI-NTA column.

Source

synthesized

Usage and Biology

The main function of the Trim21 which is E3 ligase from the TRIM family, which participate in the ubiquitin-proteasome degradation cascade of misfolded proteins. the aim of using this part in our project was that it has been used in this project to target and degrade both tau and Aβ proteins which are both considered main causes of Alzheimer’s Disease pathogenesis.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 620
  

Dry lab

Mathematical modeling

Transcription rate and translation rate under T7 promotor

the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) beside with the estimated results from the wet lab.

                 Figure 1. this figure shows the results from the transcription and translation code showing the 
                             concentration of the protein compared with the wet lab results.

Enzyme activity

Rate of consumption of ubiquitin in the system

References

1- Chu, Q., Diedrich, J. K., Vaughan, J. M., Donaldson, C. J., Nunn, M. F., Lee, K. F., & Saghatelian, A. (2016). HtrA1 Proteolysis of ApoE In Vitro Is Allele Selective. Journal of the American Chemical Society, 138(30), 9473–9478. https://doi.org/10.1021/jacs.6b03463