Difference between revisions of "Part:BBa K4165001:Design"
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===Dry Lab Characterization===
 
===Dry Lab Characterization===
 
<p style=" font-weight: bold; font-size:18px;"> Optimization </p>
 
<p style=" font-weight: bold; font-size:18px;"> Optimization </p>
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 +
Firstly, we removed the last nucleotide in the original sequence from NUDT team 2020 to avoid the frame-shifting and preparing to codon optimization.
 +
 +
Also, we optimized the sequence to be expressed in E-coli.
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<p style=" font-weight: bold; font-size:18px;"> Modeling </p>
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Through our work, we modeled Trim-21 with Dockerine and cohesin to know which one of them is more stable when it binds to Trim 21, we designed them by 5 tools (Robetta-Itasser-Alphafold-Modeller-TR Rosetta)  to reach the best model.
 +
The results and literature showed that Trim21 binds to DocS is preferable to Coh2. Our top 2 models ranked 5 out of 6 according to our QA code.
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 +
  
  

Revision as of 17:51, 4 October 2022


Truncated tripartite motif-containing 21 (TRIM21)


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 514


Design Notes

The original part on NUDT 2020 parts page had an extra nucleotide at the end so we removed it in order to avoid frameshift. Also, the whole sequence was optimized according to E.Coli BL21

Dry Lab Characterization

Optimization

Firstly, we removed the last nucleotide in the original sequence from NUDT team 2020 to avoid the frame-shifting and preparing to codon optimization.

Also, we optimized the sequence to be expressed in E-coli.

Modeling

Through our work, we modeled Trim-21 with Dockerine and cohesin to know which one of them is more stable when it binds to Trim 21, we designed them by 5 tools (Robetta-Itasser-Alphafold-Modeller-TR Rosetta) to reach the best model. The results and literature showed that Trim21 binds to DocS is preferable to Coh2. Our top 2 models ranked 5 out of 6 according to our QA code.



Source

original trim is expressed in human and we acquired the sequence from NUDT iGEM team 2020 BBa_K3396007

References

1. Clift, D., McEwan, W. A., Labzin, L. I., Konieczny, V., Mogessie, B., James, L. C., & Schuh, M. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell, 171(7), 1692-1706.e18. https://doi.org/10.1016/j.cell.2017.10.033

2. D.L. Mallery, W.A. McEwan, S.R. Bidgood, G.J. Towers, C.M. Johnson, L.C. James Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21) Proc. Natl. Acad. Sci. USA, 107 (2010), pp. 19985-19990

3. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome system. Trends in cell biology, 24(6), 352. https://doi.org/10.1016/j.tcb.2013.12.003

4. L.C. James, A.H. Keeble, Z. Khan, D.A. Rhodes, J. Trowsdale Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function Proc. Natl. Acad. Sci. USA, 104 (2007), pp. 6200-

5. Zhang, Y., Li, L., Munir, M., & Qiu, H. (2018). RING-Domain E3 Ligase-Mediated Host–Virus Interactions: Orchestrating Immune Responses by the Host and Antagonizing Immune Defense by Viruses. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2018.01083