Part:BBa_K3610031

BAK1 without native signal peptide / YFP

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

The sequence of the BAK1 protein does not contain the original signal peptide sequence of the receptor (BAK-). Instead it has been replaced by the secretion signal of the alpha-Factor from S. cerevisiae.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.

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

In a first step we inserted the single fragments making up this part into a plasmid with a gentamycin-3-acetyltransferase gene and transformed E. coli (DH10alpha) with the plasmids for amplification. In the next step we assembled the fragments in a plasmid with a spectinomycin acetyltransferase and amplified the plasmids again in the same E. coli strain. For this step we applied the techniques of Golden Gate Cloning to get the fragments in the right order into the plasmid. The restriction enzyme we chose was BsaI. For expressing this part consisting of YFP and the receptor protein, we initially intended to use promoters of different strength to get more quantitative data. Finally, we got the construct in a plasmid with a truncated version of the ADH1 promoter from S. cerevisiae. For termination, this part has the terminator sequence of the enolase 2 protein from S. cerevisiae. The plasmid also contained the TRP1 gene, which encodes phosphoribosylanthranilate isomerase, an enzyme that catalyzes the third step in tryptophan biosynthesis. This enabled us to use the same plasmid for expression in S. cerevisiae. We prepared a medium containing YNB and free amino acids, without tryptophan. S. cerevisiae cells (AP4) were transfected with the plasmid and then plated on the selective medium.

After successful transformation of S. cerevisiae cells, we checked for expression of the protein under a confocal microscope. If expression of YFP (λEx = 515 nm, λEx = 528 nm) can clearly be observed, it is reasonable to assume that the receptor domain is expressed as well, as the YFP is fused to the receptor protein. Expression of the construct was confirmed. We failed, however, to confirm localization at the cell membrane.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1758
  Illegal PstI site found at 848
  Illegal PstI site found at 893
  Illegal PstI site found at 1183
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1758
  Illegal PstI site found at 848
  Illegal PstI site found at 893
  Illegal PstI site found at 1183
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1758
  Illegal PstI site found at 848
  Illegal PstI site found at 893
  Illegal PstI site found at 1183
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1758
  Illegal PstI site found at 848
  Illegal PstI site found at 893
  Illegal PstI site found at 1183
  
• 1000
  COMPATIBLE WITH RFC[1000]