Part:BBa_K2036005

PP2CA,a type of protein from subfamily of type 2C protein phosphatases

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. As one of important part in ABA signaling network , PP2CA can form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase. Also it can directly dephosphorylate the ABFs transcription factors. These two functions both lead to inactivation of the Snf1-related(SnRK)2 clade of protein kinases, and inactivation of their downstream targets such as ABA-response element binding basic leucine zipper (bZIP) transcription factors.
Application in project: we apply PP2CA into our eukaryotic filter .When pulse OFF appears,PP2CA can dephosphorylate ABF2 and inhibite the function of SnRK2.2,these both block the expression of genes downstream the rd29A promoter.

Fig1:Eukaryote version of Signal Filter

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 90
  Illegal BamHI site found at 661
  Illegal BamHI site found at 806
  Illegal BamHI site found at 918
  Illegal XhoI site found at 65
  Illegal XhoI site found at 875
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 48
  
• 1000
  COMPATIBLE WITH RFC[1000]

Protein expression

Pichia pastoris are typical Eukaryotic expression host, but expressing plant genes are still a big challenge.So we firstly tested whether our three interest genes can be efficient expressed. We measured the secretion expression of PP2CA, ABF2 and SnRK2.2. We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinant proteins(Fig1). Compared to vector trasnfected cells, three kinds of recombinant protein plasmids trasnfected cells all expressed proteins at ~80kD under induction. The shift of protein bands suggested potential glycosylation modification.

Fig2: We analyzed our protein by SDS-PAGE，this picture is our result. From left to right,DNA marker,Wild type GS115,Wild type GS115,vector,PP2CA,ABF2,SnRK2.2.

Bi-stable function:

We constructed expression plasmid and submitted this part BBa_K2036030 to the registry.But due to the limited time,its function characterization is still under testing.However,our modeling simulation showed promising switch functions.See to Eukaryote circuit modeling.