Difference between revisions of "Part:BBa K2036003"
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<h2>Protein expression</h2>
 
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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 secretionexpression of PP2CA, ABF2 and SnRK2.2. We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinantproteins(Fig2). Compared to vector trasnfected cells, 3 kinds of recombinantprotein plasmids trasnfected cells all expressed proteins at ~80kD under induction. The shift of protein bands suggested potential glycosylation modification.
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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 recombinantproteins(Fig2). Compared to vector trasnfected cells, 3 kinds of recombinantprotein plasmids trasnfected cells all expressed proteins at ~80kD under induction. The shift of protein bands suggested potential glycosylation modification.
 
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Revision as of 06:03, 25 October 2016


SnRK2.2,SNF1-related protein kinase 2.2

Together with SnRK2.2, key component and activator of the abscisic acid (ABA) signaling pathway that regulates numerous ABA responses, such as seed germination, Pro accumulation, root growth inhibition, dormancy and seedling growth, and, to a lesser extent, stomatal closure.

We apply SnRK2.2 into our eukaryotic filter. In our system, when signal on is present, SnRK2.2 express and phosphorylate ABF2 to activate the promoter RD29A form positive feedback. At the same time, ABF2 triggers the stable production of target gene.

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 BamHI site found at 3
    Illegal BamHI site found at 660
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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 recombinantproteins(Fig2). Compared to vector trasnfected cells, 3 kinds of recombinantprotein 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_K203601330 to the registry.But due to the limited time,its function characterization is still under testing.However,our modeling simulationshowed promising switch functions.See to Eukaryote circuit modeling