Composite

Part:BBa_K3610034

Designed by: Jonas Sebastian Trottmann   Group: iGEM20_UZurich   (2020-10-05)
Revision as of 11:30, 20 October 2020 by Jtrott (Talk | contribs)


BAK1 ectodomain / mCherry N-terminal

This part contains the sequence for the ectodomain of the plant surface receptor BAK1 from A. thaliana fused to the N-terminal part of a split-mCherry protein. Additionally, instead of the signal peptide native to the plant receptor, there is the secretion signal of the alpha factor from yeast at the N-terminal domain of the receptor, replacing the original signal sequence.

Biology and Usage

BAK1

The BRI1-associated receptor kinase (BAK1) is a leucine-rich repeat receptor kinase (LRR-RK) which interacts with multiple other LRR-RKs with different functions in hormone signalling and defense response. BAK1 localizes at the plasma membrane and the endosome. The BAK1 protein forms a structure with an extracellular domain with leucine-rich repeats, a single pass transmembrane domain and an intracellular domain with a kinase function.

Among others, BAK1 interacts with the LRR-RKs EF-Tu receptor (EFR), Flagellin sensing 2 (FLS2) and cold-shock protein receptor (CORE), all of which are pathogen recognition receptors (PRR) in brassicaceae plants. Upon binding of a microbe-associated molecular pattern at the LRR domain of the PRR, BAK1 forms a heterodimer with the PRR which triggers a phosphorylation cascade, leading to upregulation of defense mechanisms.

Usage with split-mCherry

In this case, the C-terminal domain of BAK1, entailing the intracellular kinase domain, was removed from the sequence. Instead, the N-terminal domain of the split mCherry was fused to the C-terminal domain via a 15 amino acid linker.

Interaction between the BAK1, which is a coreceptor to many other PRRs, is driven by the extracellular ligand-binding domain, further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain. Coexpressed with, for example, Part:BBa_K3610040, which is the PRR EFR that contains the C-terminal domain of the split-mCherry protein instead of the intracellular kinase domain, elf18-induced interaction between BAK1 and EFR is driving the reassembly of the C-terminal and N-terminal domain of the split-mCherry, reconstituting its functionality as a fluorescent protein. In our project, we aimed at visualization of the ligand-dependent interaction between BAK1 and the respective plant PRR. This part has the potential to, in coordination with different PRRs, test for the presence of the epitopes which are recognized by the plant receptors.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 849
    Illegal PstI site found at 894
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 849
    Illegal PstI site found at 894
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 849
    Illegal PstI site found at 894
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 849
    Illegal PstI site found at 894
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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