Line 105: | Line 105: | ||
<ul> | <ul> | ||
<li>Three light inducible split proteases were designed and shown to rapidly | <li>Three light inducible split proteases were designed and shown to rapidly | ||
− | respond to | + | respond to stimulation with blue light. |
</ul> | </ul> | ||
</b></p> | </b></p> | ||
Line 115: | Line 115: | ||
activation of different biological processes. Small molecules and other chemical | activation of different biological processes. Small molecules and other chemical | ||
signals lack spatial resolution and their temporal resolution is limited by the time | signals lack spatial resolution and their temporal resolution is limited by the time | ||
− | required for | + | required for cell permeation. In comparison, induction by light as developed by |
− | + | optogenetics offers many advantages. It is fast as well as inexpensive and allows | |
for excellent spatial, temporal and dose-dependent control. | for excellent spatial, temporal and dose-dependent control. | ||
</p> | </p> | ||
Line 127: | Line 127: | ||
<p> | <p> | ||
There is a plethora of various light inducible systems available; however, not | There is a plethora of various light inducible systems available; however, not | ||
− | many are applicable to our purpose. Red light induced systems like | + | many are applicable to our purpose. Red light induced systems like the PhyB/PIF6 system |
require an | require an | ||
additional phytochrome | additional phytochrome | ||
Line 136: | Line 136: | ||
the system can only be induced once | the system can only be induced once | ||
<x-ref>Niopek2014</x-ref> | <x-ref>Niopek2014</x-ref> | ||
− | . | + | . Therefore, we selected systems responsive to blue light for the purpose of our iGEM project. |
− | + | Of these, the FKF1/GIGANTEA system displays slow association and dissociation rates | |
− | Of these, FKF1/GIGANTEA displays slow association and dissociation rates | + | |
<x-ref>Guntas2015</x-ref> | <x-ref>Guntas2015</x-ref> | ||
, making it impractical for fast response purposes, while VVD displays | , making it impractical for fast response purposes, while VVD displays | ||
Line 155: | Line 154: | ||
and in mammalian cells by <a | and in mammalian cells by <a | ||
href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Freiburg_2014</a>. | href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Freiburg_2014</a>. | ||
− | + | AsLOV2 is a small photosensory domain from <i>Avena sativa</i> | |
phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds | phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds | ||
upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin | upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin | ||
− | + | signalling. | |
</p> | </p> | ||
Line 185: | Line 184: | ||
<h3 style="clear:both;"><span id="lov" class="section colorize"> </span>LOVpep and ePDZb | <h3 style="clear:both;"><span id="lov" class="section colorize"> </span>LOVpep and ePDZb | ||
</h3> | </h3> | ||
− | <p>For initial testing and characterization of the system, we fused | + | <p>For initial testing and characterization of the system, we fused LOVpep and ePDZb |
<x-ref>Muller2014</x-ref> | <x-ref>Muller2014</x-ref> | ||
to corresponding segments of the | to corresponding segments of the | ||
<a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cle">split | <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cle">split | ||
− | firefly luciferase</a>. We tested different positions of the split protein on | + | firefly luciferase</a> (<ref>1</ref>). We tested different positions of the split protein on |
the PDZ domain, | the PDZ domain, | ||
while the split protein was kept at the N-terminus of the LOVpep domain due to the | while the split protein was kept at the N-terminus of the LOVpep domain due to the | ||
− | importance of the C-terminal peptide epitope ( | + | importance of the C-terminal peptide epitope. We also tested different ratios of both parts of the dimerizing system to determine the optimal conditions for further experiments (<ref>2</ref>B). |
− | + | ||
− | + | ||
</p> | </p> | ||
<div style="margin-left:auto; margin-right:auto; width:70%"> | <div style="margin-left:auto; margin-right:auto; width:70%"> | ||
<figure data-ref="1"> | <figure data-ref="1"> | ||
<img src="https://static.igem.org/mediawiki/2016/3/39/T--Slovenia--S.4.8.1.png"> | <img src="https://static.igem.org/mediawiki/2016/3/39/T--Slovenia--S.4.8.1.png"> | ||
− | <figcaption><b>LOVpep/ePDZ light inducible system fused to the split firefly | + | <figcaption><b>The LOVpep/ePDZ light inducible system fused to the split firefly |
luciferase.</b><br/></figcaption> | luciferase.</b><br/></figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
− | + | ||
− | + | <p>As luciferase activity was highest with split luciferase on the N-terminus of the | |
− | + | ePDZ domain and a 1:3 ratio of the LOVpep:ePDZ constructs, all subsequent experiments were | |
− | <p>As | + | performed with this conditions. An important feature for real life applications is the |
− | ePDZ domain and a 1:3 ratio of LOVpep:ePDZ, all subsequent experiments were | + | |
− | performed with this | + | |
ability of the system to be stimulated multiple times. Therefore, repeated | ability of the system to be stimulated multiple times. Therefore, repeated | ||
association | association | ||
− | and dissociation was tested in | + | and dissociation was tested in real time, by adding luciferin to the medium and |
− | measuring | + | measuring luciferase activity upon induction by light ( |
<ref>2</ref> | <ref>2</ref> | ||
− | C). The system | + | C). The system exhibited |
a delayed, but successful induction the first time, but the second induction was | a delayed, but successful induction the first time, but the second induction was | ||
− | much weaker. | + | much weaker. These results indicate that the LOVpep/ePDZ system in this setup could |
not be | not be | ||
− | induced more than once, so we decided to test | + | induced more than once, so we decided to test an additional system. |
</p> | </p> | ||
<div style="float:left; width:100%"> | <div style="float:left; width:100%"> | ||
<figure data-ref="2"> | <figure data-ref="2"> | ||
<img src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--4.9.1.png"> | <img src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--4.9.1.png"> | ||
− | <figcaption><b>LOVpep/ePDZ photoreceptors linked to split luciferase | + | <figcaption><b>LOVpep/ePDZ photoreceptors linked to the split luciferase |
reporter.</b><br/> | reporter.</b><br/> | ||
<p style="text-align:justify">(A) Schematic representation of the | <p style="text-align:justify">(A) Schematic representation of the | ||
− | light-inducible interaction between proteins containing ePDZ and LOVpep | + | light-inducible interaction between proteins containing the ePDZ and LOVpep |
domains. (B) Light inducible reporter with split | domains. (B) Light inducible reporter with split | ||
− | luciferase at the N-terminus of ePDZ domain (nLuc:ePDZb) responded to | + | luciferase at the N-terminus of the ePDZ domain (nLuc:ePDZb) responded to |
− | light more efficiently than ePDZ:nLuc. | + | light more efficiently than ePDZ:nLuc. Corresponding schematic representations of |
different arrangements | different arrangements | ||
− | of ePDZ to split firefly luciferase (nLuc). After induction the cells | + | of ePDZ fused to the N-terminal segment of split firefly luciferase (nLuc) are shown above the graph. After induction with blue light at 460nm the cells |
transfected with LOVpep/ePDZ reporter system were lysed and | transfected with LOVpep/ePDZ reporter system were lysed and | ||
− | + | luciferase activity was determined | |
− | with dual luciferase assay. ( | + | with the dual luciferase assay. (C) The LOVpep/ePDZ system responded to light stimulation |
only once. Following the addition of luciferin to the medium the cells | only once. Following the addition of luciferin to the medium the cells | ||
− | were induced | + | were induced or |
left in the dark for indicated periods. | left in the dark for indicated periods. | ||
</p> | </p> | ||
Line 248: | Line 243: | ||
<p>As it has previously been shown on the example of split Cre recombinase | <p>As it has previously been shown on the example of split Cre recombinase | ||
<x-ref>Kennedy2010</x-ref> | <x-ref>Kennedy2010</x-ref> | ||
− | the CRY2 | + | the CRY2/CIB1 interaction upon excitation with blue light seems to be a suitable tool for the |
reconstitution of split proteins, allowing for spatial, temporal and dose-dependent | reconstitution of split proteins, allowing for spatial, temporal and dose-dependent | ||
− | optical control of protein dimerization. CRY2 system has also been | + | optical control of protein dimerization. The CRY2/CIB1 system has also been used before at |
iGEM: | iGEM: | ||
<a href="https://2012.igem.org/Team:Duke/Project">Duke 2012</a>, <a | <a href="https://2012.igem.org/Team:Duke/Project">Duke 2012</a>, <a | ||
Line 264: | Line 259: | ||
<div class="content"> | <div class="content"> | ||
<p> | <p> | ||
− | CRY2 is a cryptochrome, originating from Arabidopsis thaliana and is a blue | + | CRY2 is a cryptochrome, originating from <i>Arabidopsis thaliana</i> and is a blue |
light–absorbing photosensor that binds a helix-loop-helix DNA-binding | light–absorbing photosensor that binds a helix-loop-helix DNA-binding | ||
protein CIB1 in | protein CIB1 in | ||
Line 306: | Line 301: | ||
<figure data-ref="4"> | <figure data-ref="4"> | ||
<img src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--4.9.2.png"> | <img src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--4.9.2.png"> | ||
− | <figcaption><b>CRY2PHR/CIBN light inducible receptor | + | <figcaption><b>CRY2PHR/CIBN light inducible receptor fused to split |
− | luciferase | + | luciferase responded to light efficiently and repeatedly.</b><br/> |
− | <p style="text-align:justify">(A) Response to light depended on | + | <p style="text-align:justify">(A) Response to light depended on the |
− | + | amount of the CIBN:cLuc plasmid. After induction the cells transfected with | |
− | CIBN:cLuc and CRY2PHR:nLuc were lysed and | + | the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids were lysed and luciferase activity was determined |
− | with dual luciferase assay. | + | with the dual luciferase assay. |
(B) CRY2PHR light reporter was induced repeatedly. Following the | (B) CRY2PHR light reporter was induced repeatedly. Following the | ||
− | addition of luciferin to the medium | + | addition of luciferin to the medium, cells transfected with |
− | + | the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids | |
− | (ratio 1:3) were induced and left in the dark for indicated periods. | + | (ratio 1:3) were induced with blue light at 460nm and left in the dark for indicated periods. |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
− | <p>As | + | <p>As luciferase activity was highest with a 1:3 ratio of CRY2PHR:CIBN constructs ( |
<ref>4</ref> | <ref>4</ref> | ||
A), all subsequent experiments were performed with this ratio. Next we tested if | A), all subsequent experiments were performed with this ratio. Next we tested if | ||
− | this system could be induced repeatedly in real time. The CRY2PHR/CIBN system | + | this system could be induced repeatedly in real time. The CRY2PHR/CIBN system showed a |
− | maximum activity after 2 minutes of induction | + | maximum activity after 2 minutes of induction and dropped to background 10 minutes |
after | after | ||
− | the stimulus | + | the stimulus was removed. The system could be induced repeatedly and reach high levels of activation at each stimulation ( |
<ref>4</ref> | <ref>4</ref> | ||
B). | B). | ||
</p> | </p> | ||
− | + | <figure data-ref="5"> | |
− | + | ||
− | + | ||
<img src="https://static.igem.org/mediawiki/2016/9/92/T--Slovenia--4.9.3.png"> | <img src="https://static.igem.org/mediawiki/2016/9/92/T--Slovenia--4.9.3.png"> | ||
− | <figcaption><b> | + | <figcaption><b>The CRY2PHR/CIBN mediates reconstitution of orthogonal split TEV protease upon illumination with blue light.</b><br/> |
− | + | <p style="text-align:justify">(A) Schematic representation of the CRY2PHR/CIBN light-inducible system with split protease. In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light results in heterodimerization and reconstitution of split protease (right), which in turn cleaves the cyclic luciferase reporter, resulting in increased luciferase activity. HEK293T cells were transfected with 1:3 | |
− | <p style="text-align:justify">(A) Schematic representation of CRY2PHR/ | + | ratio of plasmids coding for CRY2PHR:CIBN with split TEV protease (B) or split |
− | + | PPV protease (C). 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. The cells were lysed and luciferase activity was determined with the dual luciferase assay. Upon illumination with blue light, the protease cleaves only the cyclic reporter with the correct cleavage site (red bars), while the cyclic reporter with the mismatched cleavage site remains uncleaved (white bars). | |
− | + | ||
− | ratio of plasmids coding for CRY2PHR:CIBN with split | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
Line 372: | Line 358: | ||
<div style="float:left; width:100%"><span class="section colorize" id="cleavable luciferase"></span> | <div style="float:left; width:100%"><span class="section colorize" id="cleavable luciferase"></span> | ||
− | + | <div style="float:left; width:100%"> | |
+ | |||
+ | |||
+ | <figure data-ref="6"> | ||
<img | <img | ||
src="https://static.igem.org/mediawiki/2016/4/44/T--Slovenia--4.9.4.png"> | src="https://static.igem.org/mediawiki/2016/4/44/T--Slovenia--4.9.4.png"> |
Revision as of 13:30, 19 October 2016
nbsp;Light-dependent input signal and proteases - in progress TL
In the recent years, light has been extensively explored as a trigger signal for activation of different biological processes. Small molecules and other chemical signals lack spatial resolution and their temporal resolution is limited by the time required for cell permeation. In comparison, induction by light as developed by optogenetics offers many advantages. It is fast as well as inexpensive and allows for excellent spatial, temporal and dose-dependent control.
There is a plethora of various light inducible systems available; however, not
many are applicable to our purpose. Red light induced systems like the PhyB/PIF6 system
require an
additional phytochrome
Initially we decided to test the LOVpep/ePDZ system. This system has been used
previously at iGEM, by
EPF_Lausanne 2009,
Rutgers 2011 and Rutgers 2012
and in mammalian cells by Freiburg_2014.
AsLOV2 is a small photosensory domain from Avena sativa
phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds
upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin
signalling.
A photosensor has been prepared by engineering the AsLOV2 domain to contain a
peptide epitope SSADTWV on the C-terminus of the Jα helix (LOVpep), binding an
engineered
Erbin PDZ domain (ePDZ) upon blue light stimulation
nbsp;Results
LOVpep and ePDZb
For initial testing and characterization of the system, we fused LOVpep and ePDZb
As luciferase activity was highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of the LOVpep:ePDZ constructs, all subsequent experiments were performed with this conditions. An important feature for real life applications is the ability of the system to be stimulated multiple times. Therefore, repeated association and dissociation was tested in real time, by adding luciferin to the medium and measuring luciferase activity upon induction by light ( 2 C). The system exhibited a delayed, but successful induction the first time, but the second induction was much weaker. These results indicate that the LOVpep/ePDZ system in this setup could not be induced more than once, so we decided to test an additional system.
CRY2-CIB1
As it has previously been shown on the example of split Cre recombinase
CRY2 is a cryptochrome, originating from Arabidopsis thaliana and is a blue
light–absorbing photosensor that binds a helix-loop-helix DNA-binding
protein CIB1 in
its photoexcited state. In our system, we used the conserved N-terminal
photolyase homology region of CRY2 (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
We adapted this system for the reconstitution of split luciferase to create a blue-light sensor, which enables easy characterization for further experiments. The N- and C-terminal split fragments of the firefly luciferase were fused to the C-terminus of the CRY2PHR and the CIBN proteins, since this topology has previously been shown to work with the Cre recombinase.
As luciferase activity was highest with a 1:3 ratio of CRY2PHR:CIBN constructs ( 4 A), all subsequent experiments were performed with this ratio. Next we tested if this system could be induced repeatedly in real time. The CRY2PHR/CIBN system showed a maximum activity after 2 minutes of induction and dropped to background 10 minutes after the stimulus was removed. The system could be induced repeatedly and reach high levels of activation at each stimulation ( 4 B).
Light inducible protease
To implement light as one of the input signals for protease-based signaling pathway or logic functions, we fused the CRY2PHR and the CIBN domains to the N- and C-terminal split domains of 3 different proteases (TEVp, TEVpE and PPVp) ( 5 A) and tested their activity and orthogonality with bioluminescence assays ( 5 B) and western blotting ( 5 C). The bioluminescence assay was based on the split firefly luciferase with cleavage site for either TEVp, TEVpE or PPVp. Cleavage of this reporter results in the luciferase activity reconstitution and thus an increase in the luminescence.
The reporter used for the western blot analysis was the luciferase reporter with the appropriate cleavage substrate inserted at a permissible site and an AU1 tag at the N-terminal. Uncleaved luciferase appears as a single band on a western blot, while partial cleavage of luciferase results in two bands (uncleaved at 65 kDa and cleaved at 55 kDa) and complete cleavage results in only the smaller band ( 6 ). We showed that substrates carrying specific target peptide for proteases were cleaved after induction of split protease reporters.
Both methods showed a successful, fast and dose-dependent response. This is the first time split TEV protease has been shown to work as a light inducible system. Also this is the first time TEVpE and PPVp were prepared as split proteins and shown to function in an inducible system.