Difference between revisions of "Part:BBa K1965007"

 
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This part is one of the two components of the dimeryzing system which is induced by blue light. We adapted this system for the reconstitution of split luciferase to create a blue-light sensor. To the C-terminus of CRY, N-terminus of split firefly luciferase was added. Upon induction with light and concequent dimerisation, the split luciferase is reconstituted and regains activity, which results in biolumiescence if luciferin is added.
 
This part is one of the two components of the dimeryzing system which is induced by blue light. We adapted this system for the reconstitution of split luciferase to create a blue-light sensor. To the C-terminus of CRY, N-terminus of split firefly luciferase was added. Upon induction with light and concequent dimerisation, the split luciferase is reconstituted and regains activity, which results in biolumiescence if luciferin is added.
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<h3>Introduction </h3>
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<p>This part contains the coding sequence for the conserved N-terminal photolyase homology region of the CRY2 protein
 +
    from <i>Arabidopsis thaliana </i> (CRY2PHR; aa 1-498), fused to the N-terminal part of split firefly luciferase.
 +
    This part works together with BBa_K1965008 (CIBN:cLuc) and is one of the two components of the blue light-inducible
 +
    dimerization system CRY2/CIB1. For our project, we used the conserved N-terminal photolyase homology region of the
 +
    CRY2 protein from<i> Arabidopsis thaliana</i> (CRY2PHR; aa 1-498) that mediates light-responsiveness and the
 +
    truncated version of the CIB1 protein (CIBN; aa 1-170) without the helix-loop-helix region, which mediates DNA
 +
    binding <sup>[1]</sup>. Dimerization of CRY2PHR and CIBN can be induced by blue light illumination at approximately
 +
    460 nm, after which the interaction occurs on a millisecond timescale and can be reversed within minutes by removing
 +
    the stimulus. We adapted this system for the reconstitution of split luciferase to create a blue-light sensor. </p>
 +
<p>Upon induction with light and consequent dimerization of CIBN:cLuc (BBa_K1965008) and CRY2PHR:nLuc (BBa_K1965007),
 +
    the split luciferase is reconstituted, resulting in bioluminescence (
 +
    <ref>1</ref>
 +
    ).
 +
</p>
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</p>
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<p></p>
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<div>
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    <figure data-ref="1">
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        <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/e/ed/T--Slovenia--BBa_K1965008_2.png">
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        <figcaption><b> Figure 1: Reconstitution of split firefly luciferase upon stimulation with blue light.</b><br/>
 +
            In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light
 +
            results in heterodimerization and in turn reconstitution of split firefly luciferase (right).
 +
 +
        </figcaption>
 +
    </figure>
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</div>
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 +
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<h3>Characterization</h3>
 +
<p>To determine the most efficient ratios of both proteins, the cells were transfected with different amounts of the
 +
    plasmid encoding CIBN:cluc, while the amount of the plasmid encoding CRY2PHR:nLuc was kept constant. 24 hours after
 +
    transfection, luciferin was added to the media and the transfected cells were induced with blue light at 460nm for
 +
    30 minutes , lysed, and luciferase activity was measured with dual luciferase assay. As the luciferase activity was
 +
    highest at a 1:3 ratio of CRY2PHR:CIBN, all subsequent experiments were performed with this ratio. </p>
 +
<p>Further characterization showed that the CRY2PHR/CIBN system shows maximum activity after only 2 minutes of induction
 +
    with blue light and drops to background levels 10 minutes after the stimulus is removed. The system could also
 +
    facilitate repeated reconstitution of the split firefly luciferase after 5 consequent illuminations
 +
    <ref>2</ref>
 +
    .
 +
</p>
 +
 +
 +
</p>
 +
<p></p>
 +
 +
<div>
 +
    <figure data-ref="2">
 +
        <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/e/ef/T--Slovenia--BBa_K1965007.PNG">
 +
        <figcaption><b> Figure 2.</b><br/> Characterization of the CRY2PHR/CIBN blue light sensor linked with split
 +
            luciferase. <br/>
 +
            </b> (A) Response to light depended on concentration of CIBN:cLuc. HEK293T cells were transfected with
 +
            different ratios of plasmids encoding CIBN:cLuc and CRY2PHR:nLuc. 24 hours after transfection, 0.3mM
 +
            luciferin was added to the media and cells were illuminated with blue light at 460nm for 30minutes. After
 +
            induction the cells were lysed and luciferase activity was measured with dual luciferase assay. <br/>
 +
            </b> (B) The CRY2PHR/CIBN system can be induced repeatedly. HEK293T cells were transfected with 1:3 ratio of
 +
            plasmids encoding CIBN:cLuc and CRY2PHR:nLuc. 24 hours after transfection, 1mM luciferin was added to the
 +
            media and cells were left in the dark or illuminated with blue light at 460nm for indicated periods. <br/>
 +
 +
            .
 +
        </figcaption>
 +
    </figure>
 +
</div>
 +
 +
<sup>[1]</sup>Kennedy, M. J. et al. Rapid blue light induction of protein interaction in living cells. Nat. Methods 7,
 +
973–975 (2010). <br>
 +
 +
</body>
 +
</html>
  
  

Revision as of 00:08, 18 October 2016


His:CRY:Myc:nLuc

This part is one of the two components of the dimeryzing system which is induced by blue light. We adapted this system for the reconstitution of split luciferase to create a blue-light sensor. To the C-terminus of CRY, N-terminus of split firefly luciferase was added. Upon induction with light and concequent dimerisation, the split luciferase is reconstituted and regains activity, which results in biolumiescence if luciferin is added.

Introduction

This part contains the coding sequence for the conserved N-terminal photolyase homology region of the CRY2 protein from Arabidopsis thaliana (CRY2PHR; aa 1-498), fused to the N-terminal part of split firefly luciferase. This part works together with BBa_K1965008 (CIBN:cLuc) and is one of the two components of the blue light-inducible dimerization system CRY2/CIB1. For our project, we used the conserved N-terminal photolyase homology region of the CRY2 protein from Arabidopsis thaliana (CRY2PHR; aa 1-498) that mediates light-responsiveness and the truncated version of the CIB1 protein (CIBN; aa 1-170) without the helix-loop-helix region, which mediates DNA binding [1]. Dimerization of CRY2PHR and CIBN can be induced by blue light illumination at approximately 460 nm, after which the interaction occurs on a millisecond timescale and can be reversed within minutes by removing the stimulus. We adapted this system for the reconstitution of split luciferase to create a blue-light sensor.

Upon induction with light and consequent dimerization of CIBN:cLuc (BBa_K1965008) and CRY2PHR:nLuc (BBa_K1965007), the split luciferase is reconstituted, resulting in bioluminescence ( 1 ).

Figure 1: Reconstitution of split firefly luciferase upon stimulation with blue light.
In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light results in heterodimerization and in turn reconstitution of split firefly luciferase (right).

Characterization

To determine the most efficient ratios of both proteins, the cells were transfected with different amounts of the plasmid encoding CIBN:cluc, while the amount of the plasmid encoding CRY2PHR:nLuc was kept constant. 24 hours after transfection, luciferin was added to the media and the transfected cells were induced with blue light at 460nm for 30 minutes , lysed, and luciferase activity was measured with dual luciferase assay. As the luciferase activity was highest at a 1:3 ratio of CRY2PHR:CIBN, all subsequent experiments were performed with this ratio.

Further characterization showed that the CRY2PHR/CIBN system shows maximum activity after only 2 minutes of induction with blue light and drops to background levels 10 minutes after the stimulus is removed. The system could also facilitate repeated reconstitution of the split firefly luciferase after 5 consequent illuminations 2 .

Figure 2.
Characterization of the CRY2PHR/CIBN blue light sensor linked with split luciferase.
(A) Response to light depended on concentration of CIBN:cLuc. HEK293T cells were transfected with different ratios of plasmids encoding CIBN:cLuc and CRY2PHR:nLuc. 24 hours after transfection, 0.3mM luciferin was added to the media and cells were illuminated with blue light at 460nm for 30minutes. After induction the cells were lysed and luciferase activity was measured with dual luciferase assay.
(B) The CRY2PHR/CIBN system can be induced repeatedly. HEK293T cells were transfected with 1:3 ratio of plasmids encoding CIBN:cLuc and CRY2PHR:nLuc. 24 hours after transfection, 1mM luciferin was added to the media and cells were left in the dark or illuminated with blue light at 460nm for indicated periods.
.
[1]Kennedy, M. J. et al. Rapid blue light induction of protein interaction in living cells. Nat. Methods 7, 973–975 (2010).


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 411
    Illegal BglII site found at 870
    Illegal BamHI site found at 1349
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1642
    Illegal NgoMIV site found at 2986
    Illegal NgoMIV site found at 3007
    Illegal AgeI site found at 295
    Illegal AgeI site found at 1024
    Illegal AgeI site found at 2710
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 647
    Illegal BsaI.rc site found at 56
    Illegal SapI.rc site found at 164
    Illegal SapI.rc site found at 2892