Difference between revisions of "Part:BBa K2036029"

 
 
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<partinfo>BBa_K2036029 short</partinfo>
 
<partinfo>BBa_K2036029 short</partinfo>
  
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Kozak-ABF2-TTADH1-prd29A-Kozak-GFP-tag
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One of the basic part of genetic circuit in the eukaryotic version of signal filter.<br>
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We construct this circuit for further building of whole eukaryotic version of signal filter. Also after assembled with pCyc, it can function as control group in verification experiment of our eukaryotic signal filter.
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[[File:T--HUST-China--Experiments-Fig4.png|thumb|center|800px|Fig1:Eukaryotic circuit characterization general view]]
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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(Fig2). 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.
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</p>
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[[File: T--HUST-China--SDS.png |thumb|800px|center|Fig2: 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])
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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 more details in our Eukaryote circuit modeling.
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</p>
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<br>
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<p>
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In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Rgistery ( [https://parts.igem.org/Part:BBa_J04500 BBa_J04500]
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) to characterize the degradation tag LVAssrA.
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We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.
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</p>
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<br>
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[[File:T--HUST-China--Experiments-LVAssrA.png|thumb|800px|center|Fig3: LVAssrAtag degradation rate measurement under plac]]
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<p>
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From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.
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</p>
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<br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 06:37, 25 October 2016


Kozak-ABF2-TTADH1-prd29A-Kozak-GFP-LVAssrAtag

One of the basic part of genetic circuit in the eukaryotic version of signal filter.
We construct this circuit for further building of whole eukaryotic version of signal filter. Also after assembled with pCyc, it can function as control group in verification experiment of our eukaryotic signal filter.

Fig1:Eukaryotic circuit characterization general view


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(Fig2). 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: 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. See more details in our Eukaryote circuit modeling.


In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Rgistery ( BBa_J04500 ) to characterize the degradation tag LVAssrA. We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.


Fig3: LVAssrAtag degradation rate measurement under plac

From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.


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 321
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1765
    Illegal BsaI.rc site found at 2436