Part:BBa_K4275006:Design
TrEGIII-t
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 322
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 194
Design Notes
1. The catalytic domain and the dockerin domain is interspaced with a glycine-rich linker.
2. The 79 amino acid long type-I dockerin domain is fused at the terminal of the GS linker, followed by a TEV site and a 8xhis affinity purification tag (HHHHHHHH).
3. DNA sequence is codon-optimized based on the codon-usage table of E.coli Strain K12.MG1655, but the protein was later expressed in K. marxianus due to the lack of post-translational modifications and the formation of inclusion bodies in E.coli BL21(DE3).
4. A CBM(cellulose-binding module) domain is fused to the N terminal of the protein prior to the GH5 catalytic domain (Figure 1). This modification increases the affinity of TrEGIII to the cellulose fibres and enhance the catalytic efficiency.
Source
Trichoderma reesei ATCC 13631
Clostridium thermocellum (Dockerin-I domain)
References
1. Chang, Jui-Jen et al. "PGASO: A Synthetic Biology Tool For Engineering A Cellulolytic Yeast". Biotechnology For Biofuels, vol 5, no. 1, 2012. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-5-53.