Part:BBa_K3610033

BAK1 ectodomain with YFP - for C. reinhardtii

This part contains the ectodomain of the plant cell surface receptor from A. thaliana. I is codon optimized for expression in C. reinhardtii, to a yellow fluorescent protein. This part lacks the natural N-terminal signal sequence but instead uses the signal sequence from the possible cell wall protein, SAD1p, from C. reinhardtii.

Usage and Biology

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.

In this sequence, the C-terminal domain entailing the intracellular kinase domain was replaced with the sequence coding for the yellow fluorescent protein venus, while the ectodomain and the transmembrane domain, including the juxtamembrane domain were kept. Additionally, a signal sequence native to C. reinhardtii was fused to the N-terminal sequence, which does not contain the native signal peptide. This way, the protein can be integrated into the membrane during translation and the expression can be observed as with the receptor protein, the YFP (Exλ : 515 nm, Emλ : 528 nm) gets translated as well.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 93
  Illegal NheI site found at 925
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 855
  
• 1000
  COMPATIBLE WITH RFC[1000]