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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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
WetLab Results
Transformation of His Trim21 in BL-21 using pGS-21a vector
Figure 2. Transformed plate of His Trim21 + pGS-21a
Transformation of His Trim21 in DH-5 alpha using pJET vector
Figure 3. Transformed plate of His Trim21 + pJET
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
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