Part:BBa_K3610030

BAK1/YFP

This part can be used for expressing the plant pattern recognition receptor BRI1-associated receptro kinase (BAK1) from Arabidopsis thaliana in S. cerevisiae.

To make expression of BAK1 visible and to test observe the localization in the cell, the BAK1 coding region has been fused to a yellow fluorescent protein by a 15 amino-acid long linker.

For initiation of the expression the promoter of this part is a truncated version of the ADH1 promoter from S. cerevisiae. For termination this part has terminator sequence of the enolase 2 protein from S. cerevisiae.

Usage and Biology

BAK1 is a cell surface receptor protein with an intracellular kinase domain and an extracellular ligand binding domain. The receptor is necessary for many functions in the plant like brassinosteroid signalling and it is also a critical player in plant immunity, as BAK1 interacts with many important cell surface receptors which perceive pathogen-associated molecular patterns (PAMPs). One example of these PAMPs is the 22-amino-acid peptide flg22 from flagellin which is recognized by the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2). Upon recognizing the flg22 peptide, FLS2 interacts with BAK1. This interaction drives the immune response of the plant.

In our project, we used this part to test the expression of the whole length receptor in S. cerevisiae, as well as to observe the localization of the protein within the cell. It is important to note that the protein coding domain of this part has its original signal sequence from A. thaliana.

Characterization

Expression of BAK1/YFP in S. cerevisiae

After successful transformation of yeast cells we checked for expression of the protein under a confocal microscope. For initiation of the expression the promoter of this part is a truncated version of the ADH1 promoter from S. cerevisiae. For termination this part has terminator sequence of the enolase 2 protein from S. cerevisiae.

The results from the confocal microscopy only showed very weak fluorescence.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1560
  Illegal PstI site found at 650
  Illegal PstI site found at 695
  Illegal PstI site found at 985
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1560
  Illegal PstI site found at 650
  Illegal PstI site found at 695
  Illegal PstI site found at 985
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1560
  Illegal PstI site found at 650
  Illegal PstI site found at 695
  Illegal PstI site found at 985
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1560
  Illegal PstI site found at 650
  Illegal PstI site found at 695
  Illegal PstI site found at 985
  
• 1000
  COMPATIBLE WITH RFC[1000]