Part:BBa_K3610032:Design
BAK1 ectodomain / YFP
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 867
Illegal PstI site found at 912 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 867
Illegal PstI site found at 912 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 867
Illegal PstI site found at 912 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 867
Illegal PstI site found at 912 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
With the native signal peptide removed, the receptor sequence cannot be tranlated in-frame from the start. The scar "ggCAC" between the singal peptide and the receptor encoding domain is important for correct translation.
Kinase domain cleaved off after amino acid 287.
The Kozak consensus sequence was added for efficient translation. The amino acid from the Kozak sequence gets cleaved off during integration into the membrane.
We were able to sequence this part, which confirmed that the sequence we used was actually the correct one.
The 15 amino acid linker was chosen because it consists of small amino acids (all Ser and Gly). There is no additional tag for Western Blotting as stains for YFP are widely available.
Source
BBa_K319005, BBa_J63003, BBa_K2809002, BBa_K3610003, BBa_K416001, BBa_J176006, BBa_K2753050
References
Chinchilla, Delphine; Zipfel, Cyril; Robatzek, Silke; Kemmerling, Birgit; Nürnberger, Thorsten; Jones, Jonathan D. G. et al. (2007): A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. In: Nature 448 (7152), S. 497–500. DOI: 10.1038/nature05999.
Macho, Alberto P.; Zipfel, Cyril (2014): Plant PRRs and the Activation of Innate Immune Signaling. In: Molecular Cell 54 (2), S. 263–272. DOI: 10.1016/j.molcel.2014.03.028.
Yan, Liming; Ma, Yuanyuan; Liu, Dan; Wei, Xiaochao; Sun, Yuna; Chen, Xiaoyue et al. (2012): Structural basis for the impact of phosphorylation on the activation of plant receptor-like kinase BAK1. In: Cell Res 22 (8), S. 1304–1308. DOI: 10.1038/cr.2012.74.