Difference between revisions of "Part:BBa K3610058"

Latest revision as of 01:29, 28 October 2020

BAK1 ectodomain / LargeBit for C. reinhardtii

This part contains the sequence for the ectodomain of the plant surface receptor BAK1 fused to the LargeBit of the NanoBit system. Additionally, instead of the signal peptide native to the plant receptor, this part contains signal peptide SP7 from C. reinhardtii (Part:BBa_K3610012) at the N-terminal domain of the receptor, replacing the original signal sequence. The receptor protein coding sequence and the LargeBit are codon optimized for expression in C. reinhardtii.

Usage and Biology

BAK1

The BRI1-associated receptor kinase (BAK1) is a leucin-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 leucin-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 NanoLuc

In this case, the C-terminal domain of BAK1, entailing the intracellular kinase domain, was removed from the sequence. Instead, the LargeBit part of the split-NanoLuc protein domain 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. It is possible that dimerization of the two receptors can be induced without the intracellular kinase domain of neither the target receptor nor BAK1.
We were interested in testing whether coexpression of this part with the EFR or CORE ectodomain fused to the SmallBit domain of split-NanoLuc in C. reinhardtii would allow to visually capture the presence of the bacterial epitopes (csp22 and elf18) in water samples by ligand-dependent interaction of the two receptors, an interaction which would drive the reconstitution of the split-NanoLuc protein.

Sequence and Features