Part:BBa_K4275013:Design
OlpB-3C-Ag3
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 706
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1252
Illegal NgoMIV site found at 1341
Illegal AgeI site found at 183
Illegal AgeI site found at 319
Illegal AgeI site found at 370
Illegal AgeI site found at 502
Illegal AgeI site found at 868
Illegal AgeI site found at 886
Illegal AgeI site found at 1312 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1069
Illegal SapI.rc site found at 1081
Illegal SapI.rc site found at 1705
Design Notes
1. A 6x His affinity purification tag(HHHHHH) added to N-terminal following the start codon, to allow for Ni-NTA purification.
2. The number of cohesins is cut from 7 to 3 to reduce cellular pressure, ensuring successful heterologous expression in E.coli.
3. The original anchoring domain of OlpB that binds to yeast surface, glycosylphosphatidylinositol (ScGPI), is replaced by Ag3 so that OlpB can attach to the cell surface of E.coli through the interaction between Ag3 and Nb3 cell surface expression system (BBa_K4275026).
4. The OlpB scaffoldin domain and the Ag3 antigen domain are interspaced with a 10 aa flexible GS linker GGGGSGGGGS to avoid the steric inhibtion of the giant OlpB scaffold.
5. DNA sequence is codon-optimized based on the codon-usage table of E.coli Strain K12.MG1655.
Source
Clostridium thermocellum
References
1. Anandharaj, Marimuthu et al. "Constructing A Yeast To Express The Largest Cellulosome Complex On The Cell Surface". Proceedings Of The National Academy Of Sciences, vol 117, no. 5, 2020, pp. 2385-2394. Proceedings Of The National Academy Of Sciences, https://doi.org/10.1073/pnas.1916529117.