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Part:BBa_K3610031
BAK1 without native signal peptide / YFP
This part can be used for expressing the plant pattern recognition receptor BRI1-associated receptro kinase (BAK1) from Arabidopsis thaliana in S. cerevisiae.
The sequence of the BAK1 protein does not contain the original signal peptide sequence of the receptor (BAK-). Instead it has been replaced by the secretion signal of the alpha-Factor from S. cerevisiae. To make expression of BAK1 visible and to test observe the localization in the cell, the BAK1 coding region has been fused to a yellow fluorescent protein by a 15 amino-acid long linker.
For initiation of the expression the promoter of this part is a truncated version of the ADH1 promoter from S. cerevisiae. For termination this part has terminator sequence of the enolase 2 protein from S. cerevisiae.
Usage and Biology
BAK1 is a cell surface receptor protein with an intracellular kinase domain and an extracellular ligand binding domain. The receptor is necessary for many functions in the plant like brassinosteroid signalling and it is also a critical player in plant immunity, as BAK1 interacts with many important cell surface receptors which perceive pathogen-associated molecular patterns (PAMPs). One example of these PAMPs is the 22-amino-acid peptide flg22 from flagellin which is recognized by the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2). Upon recognizing the flg22 peptide, FLS2 interacts with BAK1. This interaction drives the immune response of the plant.
In our project, we used this part to test the expression of the whole length receptor in S. cerevisiae, as well as to observe the localization of the protein within the cell. It is important to note that the protein coding domain of this part has its original signal sequence from A. thaliana.
Characterization
Expression of BAK1-/YFP in S. cerevisiae
After successful transformation of S. cerevisiae cells, we checked for expression of the protein under a confocal microscope. If expression of YFP (λEx = 515 nm, λEx = 528 nm) can clearly be observed, it is reasonable to assume that the receptor domain is expressed as well. Expression of the construct was confirmed. We failed, however, to confirm localization at the cell membrane.
Sequence and Features
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- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 1000COMPATIBLE WITH RFC[1000]
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