Difference between revisions of "Part:BBa K2036002"
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<partinfo>BBa_K2036002 short</partinfo>
 
<partinfo>BBa_K2036002 short</partinfo>
 
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<br/>
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A promoter region upstream Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments .Promoter RD29A contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences which are cis-acting elements. So the promoter RD29A can be active by transcription factor and then start downstream genes expression.
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A promoter region upstream Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments .Promoter rd29A contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences which are cis-acting elements. So the promoter rd29A can be active by transcription factor and then start downstream genes expression.
 
<br />
 
<br />
 
=== Application: ===
 
=== Application: ===
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<br> we apply RD29A promoter into our eukaryotic filter .when transcription factor ABF2 is phosphorylated by kinase SnRK2.2 ,the RD29A promoter can be activated and promote the expression of SnRK2.2 and target gene. These make the system form a positive feedback and lead to stable expression of target gene.
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<br> We apply rd29A promoter into our eukaryotic filter .When transcription factor ABF2 is phosphorylated by kinase SnRK2.2 ,the rd29A promoter can be activated and promote the expression of SnRK2.2 and target gene. These make the system form a positive feedback and lead to stable expression of target gene.
 
[[File:T--HUST-China--Description-Fig-prokaryote.png|thumb|500px|center|Fig1:Eukatyote version of Signal Filter]]
 
[[File:T--HUST-China--Description-Fig-prokaryote.png|thumb|500px|center|Fig1:Eukatyote version of Signal Filter]]
  
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<h2>Protein expression</h2>
 
<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.
+
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(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.
 
</p>
 
</p>
 
<br>
 
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<h2>Bi-stable function:</h2>
 
<h2>Bi-stable function:</h2>
 
<p>
 
<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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We constructed expression plasmid and submitted this part BBa_K2036030[https://parts.igem.org/Part:BBa_K2036030] 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.
 
</p>
 
</p>

Revision as of 05:45, 25 October 2016


Prd29A
A promoter region upstream Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments .Promoter rd29A contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences which are cis-acting elements. So the promoter rd29A can be active by transcription factor and then start downstream genes expression.

Application:


We apply rd29A promoter into our eukaryotic filter .When transcription factor ABF2 is phosphorylated by kinase SnRK2.2 ,the rd29A promoter can be activated and promote the expression of SnRK2.2 and target gene. These make the system form a positive feedback and lead to stable expression of target gene.

Fig1:Eukatyote version of Signal Filter

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 153


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(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[1] 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.