Difference between revisions of "Part:BBa K2036004"
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<partinfo>BBa_K2036004 parameters</partinfo>
 
<partinfo>BBa_K2036004 parameters</partinfo>
 
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<h2>Protein expression</h2>
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<p>
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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(Fig1). 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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</p>
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<br>
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[[File:T--HUST-China--SDS.png|800px|thumb|center|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.]]
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<h2>Bi-stable function:</h2>
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<p>
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We constructed expression plasmid and submitted this part BBa_K2036030to 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
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</p>

Revision as of 14:52, 19 October 2016


ABF2,abscisic acid responsive elements-binding factor 2

Involved in ABA and stress responses and acts as a positive component of glucose signal transduction. Functions as transcriptional activator in the ABA-inducible expression of RD29B. Binds specifically to the ABA-responsive element (ABRE) of the RD29B gene promoter.

We apply ABF2 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 production of the gene of interest.

Fig1:Eukaryote version of Signal Filter

Usage and Biology

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 309
  • 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 secretionexpression of PP2CA, ABF2 and SnRK2.2. We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinantproteins(Fig1). 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_K2036030to 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