Difference between revisions of "Part:BBa K3610040"

Revision as of 20:36, 15 October 2020

EFR ectodomain / mCherry C-terminal for S. cerevisiae

This part entails the ectodomain of the plant pattern recognition receptor EFR from A. thaliana which is fused to the C-terminal domain of the mCherry protein via a 15 amino acid linker. The C-terminal sequence of the receptor protein, encoding the signal peptide, was removed from the sequence. To ensure localization, the secretion signal of the alpha Factor from yeast is added instead.

Usage and Biology

EFR

Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants firts defence mechanism against potential pathogens. The EFR receptor is also a leucin-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucin-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.

Usage with mCherry

In this case, the C-terminal domain of EFR, 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.

The ligand-dependent interaction of EFR with its coreceptor BAK1 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 of neither EFR nor BAK1. Coexpressed with Part:BBa_K3610034, which is the ectodomain of BAK1 fused to the N-terminal part of mCherry, elf18-induced interaction between BAK1 and EFR drives the reassembly of the C-terminal and N-terminal domain of the split-mCherry, reconstituting its functionality as a fluorescent protein. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs EFR and BAK1. This enables us to use this part, in coordination with Part:BBa_K3610035, to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaciton between the receptors, causing the split-mCherry parts to rejoin and generate a funcitonal fluorescent protein.

Sequence and Features