Part:BBa_K4275004:Design
CBHII-t
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 535
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 813
Illegal AgeI site found at 1111 - 1000COMPATIBLE WITH RFC[1000]
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 GH6 catalytic domain (Figure 1). This modification increases the affinity of CBHII to the cellulose fibres and enhance the catalytic efficiency.
5. A CBM(cellulose-binding module) domain is fused to the N terminal of the protein prior to the linker domain (Figure 1). This modification increases the affinity of MtCDH to the cellulose fibres and enhance the catalytic efficiency.
Source
Synthetic Gene
Clostridium thermocellum (Dockerin-I domain)
References
1. Chang, Jui-Jen et al. "Assembling A Cellulase Cocktail And A Cellodextrin Transporter Into A Yeast Host For CBP Ethanol Production". Biotechnology For Biofuels, vol 6, no. 1, 2013. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-6-19.