Difference between revisions of "Part:BBa K2510102"

 
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<partinfo>BBa_K2510102 short</partinfo>
 
<partinfo>BBa_K2510102 short</partinfo>
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Dronpa is a  reversible photoswitchable fluorescent protein that is switched on by default “fluorescent” and is switched off when illuminated by cyan light (~500nm). Dronpa Fluorescence is recovered by shining violet light (~400nm). And has been used in a design that facilitates the optical control of protein activities
  
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This part contains a device of two copies of Dronpa Fluorescent Protein that are codon optimized for E Coli with two BsaI cutting site in between to allow the insertion of various proteins to be tested . This coding sequence of the 2 dronpa domains has 2 mutations I4V and R149H in the first dronpa domain and an F78S mutation in the second domain that were obtained by error prone PCR. which makes it a better version of the part that already exists in the registry. <a href="https://parts.igem.org/Part:BBa_K1680006">here</a>
  
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This mutant version of Dronpa has showed a better performance than the wild type in controling the activity of both TetR [fig1] and β-galactosidase[fig 3], the work flow of the  β-galactosidase activity experiment is indicated in figure 2  </p>
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<img src="https://static.igem.org/mediawiki/2017/e/e2/Logic_tetR_PB.png">
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<img src="https://static.igem.org/mediawiki/2017/8/8e/Logic_P22c2_PB.png">
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<img src="https://static.igem.org/mediawiki/2017/1/19/Logic_HKcIfigure_PB.png">
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                                            <span  class="image-span text-center">
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                                            <b>Figure 1:Results of the cell-free experiment. Each promoter was tested with its cognate repressors. Top: Testing with TetR caged with either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)Middle: Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated version(BBa_K2510113).Bottom: Testing with HKcI caged with either wt-Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111)
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<img id="fig2" src="https://static.igem.org/mediawiki/2017/6/61/Aya_figure_13.png" />
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                                    <span  class="image-span text-center">
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                                            <b>Figure 2: </b>  An overview of the experiment done to evaluate the activity of β-galactosidase-Dronpa fusion.
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                                    </span>
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                            <img id="fig3" src="https://static.igem.org/mediawiki/2017/0/01/Aya_figure_14.png" />
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                                    <span  class="image-span text-center">
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                                            <b>Figure 3: </b> X-Gal grayscale picture, testing the activity of β-galactosidase fusion with both wtDronpa and mutDronpa, indicating that β-galactosidase-mutDronpa fusion is more responsive to cyan light than the β-galactosidase-wtDronp.
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                                    </span>
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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===

Revision as of 03:19, 2 November 2017


pdDronpa mutant Dronpa is a reversible photoswitchable fluorescent protein that is switched on by default “fluorescent” and is switched off when illuminated by cyan light (~500nm). Dronpa Fluorescence is recovered by shining violet light (~400nm). And has been used in a design that facilitates the optical control of protein activities This part contains a device of two copies of Dronpa Fluorescent Protein that are codon optimized for E Coli with two BsaI cutting site in between to allow the insertion of various proteins to be tested . This coding sequence of the 2 dronpa domains has 2 mutations I4V and R149H in the first dronpa domain and an F78S mutation in the second domain that were obtained by error prone PCR. which makes it a better version of the part that already exists in the registry. here This mutant version of Dronpa has showed a better performance than the wild type in controling the activity of both TetR [fig1] and β-galactosidase[fig 3], the work flow of the β-galactosidase activity experiment is indicated in figure 2

Figure 1:Results of the cell-free experiment. Each promoter was tested with its cognate repressors. Top: Testing with TetR caged with either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)Middle: Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated version(BBa_K2510113).Bottom: Testing with HKcI caged with either wt-Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111) Figure 2: An overview of the experiment done to evaluate the activity of β-galactosidase-Dronpa fusion. Figure 3: X-Gal grayscale picture, testing the activity of β-galactosidase fusion with both wtDronpa and mutDronpa, indicating that β-galactosidase-mutDronpa fusion is more responsive to cyan light than the β-galactosidase-wtDronp. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 755
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 758
    Illegal BsaI.rc site found at 752