Part:BBa_K4165202

Trim-(GGGGS)3-Docs

This parts code for the trim21 E3 ligase having his PRYSPRY domain truncated (BBa_K3396007), fused to type 1 Dockerin module derived from Clostridium thermocellum cellulosome scaffoldin using Glycine serine flexible linker repeated three times to maintain part flexibility needed during target ubiquitination.

source

Synthetic Fusion protein

Sequence and Features

sequence was optimized for E.coli expression

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 535
  

References

1-Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.

WetLab Results

Transformation of His Trim21 (L) Doc in BL-21 using pGS-21a

                                   Figure 1. Transformed plate of His Trim21 (L) Doc + pGS-21a 

Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector

                                   Figure 2. Transformed plate of His Trim21 (L) Doc + pJET 

Comparison between chemical lysis and sonication for His Trim21 (L) DOC

Figure 3. This graph shows a significant difference between chemical lysis and sonication for His Trim21 (L) DOC, after we had the results we optimized our protocol to use sonication for His Trim21 (L) DOC