Difference between revisions of "Part:BBa K3610037"

Latest revision as of 11:38, 20 October 2020

BAK1 ectodomain / mCherry C-terminal - for C. reinhardtii

This part contains the sequence for the ectodomain of the plant surface receptor BAK1 fused to the C-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 possible cell wall protein SAD1 from C. reinhardtii at the N-terminal domain of the receptor, replacing the original signal sequence. The ectodomain and the mCherry sequence are codon optimized for expression in C. reinhardtii.

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 C-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_K3610041, which is the PRR EFR that contains the N-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 expressing this part in C. reinhardtii in order to visualize the ligand-dependent interaction between BAK1 and the respective plant PRR. This enables us to use this part, in coordination with different PRRs, to test for the presence of the epitopes which are recognized by the plant receptors.

Sequence and Features