Difference between revisions of "Part:BBa K2036031"
 
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<partinfo>BBa_K2036031 short</partinfo>
 
<partinfo>BBa_K2036031 short</partinfo>
  
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PP2CA-TTADH1-pCyc<br>is one of the Basic Part in the eukaryotic version of signal filter ([https://parts.igem.org/Part:BBa_K2036030 BBa_K2036030])and it is constructed by 3A assembly.<br>
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It is one of the Basic Part in the eukaryotic version of signal filter ([https://parts.igem.org/Part:BBa_K2036030 BBa_K2036030])and it is constructed by In-Fusion method.<br>
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We construct this circuit for further building of whole eukaryotic version of signal filter and for verifying the function of related proteins.<br>
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<br>
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<h3> Protein expression</h3>
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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 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, 3 kinds of recombinant protein 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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[[File: T--HUST-China--SDS.png |thumb|800px|center|Fig1: SDS PAGE]]
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<br>
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<h3> Bi-stable function:</h3>
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<p>
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We constructed expression plasmid and submitted this part ([https://parts.igem.org/Part:BBa_K2036030 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. ([http://2016.igem.org/Team:HUST-China/Model/model-euk See more details in our Eukaryote circuit modeling]).
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 06:16, 25 October 2016


PP2CA-TTADH1-pCyc

It is one of the Basic Part in the eukaryotic version of signal filter (BBa_K2036030)and it is constructed by In-Fusion method.

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, 3 kinds of recombinant protein plasmids trasnfected cells all expressed proteins at ~80kD under induction. The shift of protein bands suggested potential glycosylation modification.

Fig1: SDS PAGE


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. ([http://2016.igem.org/Team:HUST-China/Model/model-euk See more details in our Eukaryote circuit modeling]).


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]