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+ | processEscapes: true | ||
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− | }) | + | }) |
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<i class="selected radio icon"></i> | <i class="selected radio icon"></i> | ||
− | <b> | + | <b>Project</b> |
</a> | </a> | ||
− | <a class="item" href="# | + | <a class="item" href="#achievements" style="margin-left: 10%"> |
<i class="selected radio icon"></i> | <i class="selected radio icon"></i> | ||
− | <b> | + | <b>Achievements</b> |
</a> | </a> | ||
− | <a class="item" href="# | + | <a class="item" href="#requirements" style="margin-left: 10%"> |
<i class="selected radio icon"></i> | <i class="selected radio icon"></i> | ||
− | <b> | + | <b>Medal requirements</b> |
</a> | </a> | ||
− | <a class="item" href=" | + | <a class="item" href="idea"> |
<i class="chevron circle right icon"></i> | <i class="chevron circle right icon"></i> | ||
− | <b> | + | <b>Idea</b> |
</a> | </a> | ||
</div> | </div> | ||
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<!-- content goes here --> | <!-- content goes here --> | ||
<div class="main ui citing justified container"> | <div class="main ui citing justified container"> | ||
− | < | + | <h1><span id="intro" class="section"> </span>Modeling logic gates</h1> |
+ | <div class="ui segment"> | ||
<p> | <p> | ||
Engineering and designing biological circuits constitute a central core of synthetic biology. In | Engineering and designing biological circuits constitute a central core of synthetic biology. In | ||
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<p>The designed binary logic gates can be divided into 5 subgroups, based on the position of the | <p>The designed binary logic gates can be divided into 5 subgroups, based on the position of the | ||
protease cleavage | protease cleavage | ||
− | sites: | + | sites:</p> |
<ul> | <ul> | ||
<li>a) cleavage site between coiled-coils: conjunction, disjunction and both projection | <li>a) cleavage site between coiled-coils: conjunction, disjunction and both projection | ||
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<img class="ui huge centered image" | <img class="ui huge centered image" | ||
src="https://static.igem.org/mediawiki/2016/2/20/T--Slovenia--5.5.2.png"> | src="https://static.igem.org/mediawiki/2016/2/20/T--Slovenia--5.5.2.png"> | ||
− | <figcaption>Scheme of the modelled function $f_1$.The output is represented with the emission of | + | <figcaption><b>Scheme of the modelled function $f_1$.</b>The output is represented with the |
+ | emission of | ||
light induced | light induced | ||
by | by | ||
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<img class="ui huge centered image" | <img class="ui huge centered image" | ||
src="https://static.igem.org/mediawiki/2016/c/c7/T--Slovenia--5.5.3.png"> | src="https://static.igem.org/mediawiki/2016/c/c7/T--Slovenia--5.5.3.png"> | ||
− | <figcaption>Scheme of the modelled function $f_2$. The output is represented with the emission | + | <figcaption><b>Scheme of the modelled function $f_2$.</b> The output is represented with the |
+ | emission | ||
of light induced | of light induced | ||
by | by | ||
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<img class="ui huge centered image" | <img class="ui huge centered image" | ||
src="https://static.igem.org/mediawiki/2016/c/c1/T--Slovenia--5.5.4.png"> | src="https://static.igem.org/mediawiki/2016/c/c1/T--Slovenia--5.5.4.png"> | ||
− | <figcaption>Scheme of the modelled function $f_3$. The output is represented with the emission | + | <figcaption><b>Scheme of the modelled function $f_3$.</b> The output is represented with the |
+ | emission | ||
of light induced | of light induced | ||
by | by | ||
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<img class="ui huge centered image" | <img class="ui huge centered image" | ||
src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--5.5.5.png"> | src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--5.5.5.png"> | ||
− | <figcaption>Scheme of the modelled function $f_4$. The output is represented with the emission | + | <figcaption><b>Scheme of the modelled function $f_4$.</b> The output is represented with the |
+ | emission | ||
of light induced | of light induced | ||
by | by | ||
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</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
− | + | </div> | |
<h3><span id="model" class="section"> </span>Deterministic modeling</h3> | <h3><span id="model" class="section"> </span>Deterministic modeling</h3> | ||
+ | <div class="ui segment"> | ||
We have established the following ordinary differential equations (ODEs) based model: | We have established the following ordinary differential equations (ODEs) based model: | ||
<h4>Projection function $f_1$</h4> | <h4>Projection function $f_1$</h4> | ||
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\end{align} | \end{align} | ||
− | <h4>Converse implication | + | <h4>Converse implication f<sub>3</sub></h4> |
\begin{align} | \begin{align} | ||
b'(t) =& | b'(t) =& | ||
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k'(t) =& \alpha_1- \delta_1 * k(t) - \tau * k(t) * p_1(t) - \tau * k(t) * p_2(t), \\ | k'(t) =& \alpha_1- \delta_1 * k(t) - \tau * k(t) * p_1(t) - \tau * k(t) * p_2(t), \\ | ||
k_1'(t) =& -\delta_1 * k_1(t) - \beta_1 * b(t) * k_1(t) + \gamma_2 * k_{12}(t) + \\ | k_1'(t) =& -\delta_1 * k_1(t) - \beta_1 * b(t) * k_1(t) + \gamma_2 * k_{12}(t) + \\ | ||
− | & \gamma_2 * k_{123}(t) + \beta_2 * k_1b(t) - \gamma_1 * k_1(t) * k_2(t) - | + | & \gamma_2 * k_{123}(t) + \beta_2 * k_1b(t) - \gamma_1 * k_1(t) * k_2(t) - |
− | + | \gamma_1 * k_1(t) * k_{23}(t) \\ | |
+ | &+ \tau * k(t) * p_1(t) + \tau * k_1k_2(t) * p_1(t), \\ | ||
k_{12}'(t) =& -\delta_1 * k_{12}(t) - \gamma_2 * k_{12}(t) + | k_{12}'(t) =& -\delta_1 * k_{12}(t) - \gamma_2 * k_{12}(t) + | ||
\gamma_1 * k_1(t) * k_2(t), \\ | \gamma_1 * k_1(t) * k_2(t), \\ | ||
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\end{align} | \end{align} | ||
− | <h4>Mathematical nonimplication | + | <h4>Mathematical nonimplication f<sub>4</sub></h4> |
\begin{align} | \begin{align} | ||
v'(t) =& | v'(t) =& | ||
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\tau * g_1d(t) * p_2(t), \\ | \tau * g_1d(t) * p_2(t), \\ | ||
g'(t) =& \alpha_1- \delta_1 * g(t) - \tau * g(t) * p_1(t), \\ | g'(t) =& \alpha_1- \delta_1 * g(t) - \tau * g(t) * p_1(t), \\ | ||
− | g_1'(t) =& -\delta_1 * g_1(t) - \beta_1 * d(t) * g_1(t) - | + | g_1'(t) =& -\delta_1 * g_1(t) - \beta_1 * d(t) * g_1(t) - \gamma_1 * d_1(t) * g_1(t) \\ |
− | + | & + \beta_2 * g_1d(t) + \gamma_2 * g_1d_1(t) + | |
− | + | ||
\gamma_2 * g_1g_2(t) - \gamma_1 * g_1(t) * g_2(t), \\ | \gamma_2 * g_1g_2(t) - \gamma_1 * g_1(t) * g_2(t), \\ | ||
g_1d'(t) =& | g_1d'(t) =& | ||
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<p> | <p> | ||
The function of light presence, denoted with $l(t)$, $l_1(t)$ or $l_2(t)$, is a piecewise | The function of light presence, denoted with $l(t)$, $l_1(t)$ or $l_2(t)$, is a piecewise | ||
− | function which equals | + | function which equals 1 if the light is present and 0 otherwise. Functions $p_1$, $p_2$, $g$, |
− | + | $g_1$, | |
− | + | $g_1d$, $g_1d_1$, $g_1g_2$, $g_1i$, $g_2$, $c$, $c_1$, $c_2$, $c_2d$, $cd$, $cd_2$, $w$, $z$, | |
− | + | $d$, $d_1$, $d_2$, $k$, $k_1$, $k_{12}$, | |
− | + | $k_{123}$, $k_1b$, $k_1k_2$, $k_2$, $k_{23}$, $k_3$, $i$, $b$, $k$, $v$, $u$, $w$, $z$ present | |
− | + | concentrations of the | |
− | + | equally labelled proteins. The constants used for the model are described in | |
− | $k_{123}$, $k_1b$, | + | |
− | + | ||
− | + | ||
− | labelled | + | |
− | + | ||
− | + | ||
− | + | ||
<ref>tab:refs</ref> | <ref>tab:refs</ref> | ||
. | . | ||
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</tbody> | </tbody> | ||
</table> | </table> | ||
− | + | </div> | |
<h1><span id="results" class="section"> </span>Results</h1> | <h1><span id="results" class="section"> </span>Results</h1> | ||
+ | <div class="ui segment"> | ||
<p>We simulated the dynamics of established logic gates with the numerical integration of their | <p>We simulated the dynamics of established logic gates with the numerical integration of their | ||
mathematical models | mathematical models | ||
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</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
− | |||
− | |||
− | |||
</div> | </div> | ||
</div> | </div> |
Revision as of 18:50, 17 October 2016
Modeling logic gates
Engineering and designing biological circuits constitute a central core of synthetic biology. In
the context of our
iGEM
project, one the purpose was to create, tune and regulate novel pathways in living cells using a
fast-relay system.
The
toolset of
orthogonal proteases
that we developed worked as input for logic
function in mammalian cells. Therefore, here we propose schemes for implementation of all 16
two input
binary logic functions based on a protein-protein interaction (coiled coil) and proteolysis
system in cells. Designed logic gates based on
protein-protein interaction are
expected to have a shorter time delay compared to their analogues based on genetic regulatory
networks
The main post-translational modification on which signaling and information processing systems are based is protein phosphorylation, which enables reversibility and fast response. Proteolysis is on the other hand irreversible, which imposes some limitations with respect to phosphorylation. However for many applications fast activation is most important, while the time to reset the system in the resting state is not that important.
Our protein-based system is designed in such a way that it works through coiled coil interactions, where each coiled coil in the system is either free or bound to its partner depending on the proteolytic activity. Furthermore, the signal output is represented by reconstitution of a split protein (i.e. luciferase or protease), which is fused separately to different coiled coil segments. To prove the feasibility of this design, we simulated the system's behavior using deterministic modelling. The simulations were run in Wolfram Mathematica, using xCellerator's xlr8r libraries.
The designed binary logic gates can be divided into 5 subgroups, based on the position of the protease cleavage sites:
- a) cleavage site between coiled-coils: conjunction, disjunction and both projection functions;
- b) cleavage site between the coiled-coil and split protease: logical NAND, logical NOR and both negations;
- c) cleavage sites between coiled-coils as well as between the coiled-coil and split protease in the same construct: material implication and converse implication;
- d) cleavage sites between coiled-coils as well as between the coiled-coil and split protease in different constructs: exclusive disjunction, logical biconditional, material nonimplication and converse nonimplication;
- e) no cleavage sites: tautology and contradiction.
For applications that require fast response (e.g. protein secretion), which are the purpose of our attempt, only falsity preserving gates are appropriate, as biological systems usually require fast activation and not fast deactivation. The following functions correspond to the desired condition: both projection functions, conjunction, disjunction, exclusive disjunction, material nonimplication, converse nonimplication and true.
Since the dynamics of both functions in subgroup e) is trivial, i.e. output is a constant, their modelling is omitted. We selected a single function from the other four subgroups, for which a mathematical model was established and analysed. We selected the following functions $f_1(x_1, x_2) = x_1$ from subgroup a), $f_2(x_1, x_2) = \neg(x_1 \vee x_2)$ from b), $f_3(x_1, x_2) = x_2 \Rightarrow x_1$ from c) and $f_4(x_1, x_2) = \neg(x_1 \Rightarrow x_2)$ from d).
Inducible proteases were assumed as the two input variables for each function. The logical values true and false were in all the cases presented with high and low amounts of output proteins or input proteases, respectively. Where the output signal is presented with several different proteins, the sum of their concentrations was observed. The schemes of the assumed reactions included in the implementation of described logical functions are represented in fig:scheme_buffer , fig:scheme_nor , fig:schemes_imply and fig:schemes_nimply . All of them ignore the leakage due to the binding of the coiled-coils before cleavage, which could be solved by setting the building elements with appropriate parameters as demonstrated in the experimental section on the CC-based logic design.
![](https://static.igem.org/mediawiki/2016/2/20/T--Slovenia--5.5.2.png)
![](https://static.igem.org/mediawiki/2016/c/c7/T--Slovenia--5.5.3.png)
![](https://static.igem.org/mediawiki/2016/c/c1/T--Slovenia--5.5.4.png)
![](https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--5.5.5.png)
Deterministic modeling
Projection function $f_1$
\begin{align} v'(t) =& \alpha_2 - \delta_1 * v(t) - \sigma_1 * v(t) * u(t) * l(t) + \sigma_2 * p_1(t), \\ u'(t) =& \alpha_2 - \delta_1 * u(t) - \sigma_1 * v(t) * u(t) * l(t) + \sigma_2 * p_1'(t), \\ g'(t) =& \alpha_1- \delta_1 * g(t) - \tau * g(t) * p_1(t), \\ g_1'(t) =& -\delta_1 * g_1(t) + \gamma_2 * g_1g_2(t) + \beta_2 * g_1i(t) - \gamma_1 * g_1(t) * g_2(t) - \beta_1 * g_1(t) * i(t), \\ g_1g_2'(t) =& -\gamma_2 * g_1g_2(t) + \gamma_1 * g_1(t) * g_2(t) + \tau * g(t) * p_1(t), \\ g_1i'(t) =& -\delta_1 * g_1i(t) - \beta_2 * g_1i(t) + \beta_1 * g_1(t) * i(t), \\ g_2'(t) =& \gamma_2 * g_1g_2(t) - \delta_1 * g_2(t) - \gamma_1 * g_1(t) * g_2(t), \\ i'(t) =& \alpha_1+ \beta_2 * g_1i(t) - \delta_1 * i(t) - \beta_1 * g_1(t) * i(t),\\ p_1'(t) =& \sigma_1 * v(t) * u(t) * l(t) - \sigma_2 * p_1(t) \end{align}Logical NOR $f_2$
\begin{align} c'(t) =& \alpha_1- \delta_1 * c(t) + \beta_2 * cd(t) - \beta_1 * c(t) * d(t) - \tau * c(t) * p_1(t), \\ c_1'(t) =& -\delta_1 * c_1(t) + \tau * c(t) * p_1(t) + \tau * cd(t) * p_1(t), \\ c_2'(t) =& -\delta_1 * c_2(t) + \tau * c(t) * p_1(t), \\ c_2d'(t) =& \tau * cd(t) * p_1(t), \\ cd'(t) =& -\delta_1 * cd(t) - \beta_2 * cd(t) + \beta_1 * c(t) * d(t) - \tau * cd(t) * p_1(t) - \tau * cd(t) * p_2(t), \\ cd_2'(t) =& \tau * cd(t) * p_2(t), \\ v'(t) =& \alpha_2 - \delta_1 * v(t) - \sigma_1 * v(t) * u(t) * l_1(t) + \sigma_2 * p_1(t), \\ w'(t) =& \alpha_2 - \delta_1 * w(t) - \sigma_1 * w(t) * z(t) * l_2(t)+ \sigma_2 * p_2(t), \\ u'(t) =& \alpha_2 - \delta_1 * u(t) - \sigma_1 * v(t) * u(t) * l_1(t) + \sigma_2 * p_1(t), \\ z'(t) =& \alpha_2 - \delta_1 * z(t) - \sigma_1 * w(t) * z(t) * l_2(t) + \sigma_2 * p_2(t), \\ d'(t) =& \alpha_1+ \beta_2 * cd(t) - \delta_1 * d(t) - \beta_1 * c(t) * d(t) - \tau * d(t) * p_2(t), \\ d_1'(t) =& -\delta_1 * d_1(t) + \tau * cd(t) * p_2(t) + \tau * d(t) * p_2(t), \\ d_2'(t) =& -\delta_1 * d_2(t) + \tau * d(t) * p_2(t), \\ p_1'(t) =& \sigma_1 * v(t) * u(t) * l_1(t) - \sigma_2 * p_1(t), \\ p_2'(t) =& \sigma_1 * w(t) * z(t) * l_2(t) - \sigma_2 * p_2(t) \end{align}Converse implication f3
\begin{align} b'(t) =& \alpha_1- \delta_1 * b(t) - \beta_1 * b(t) * k_1(t) + \beta_2 * k_1b(t), \\ v'(t) =& \alpha_2 - \delta_1 * v(t) - \sigma_1 * v(t) * u(t) * l_1(t) + \sigma_2 * p_1(t), \\ w'(t) =& \alpha_2 - \delta_1 * w(t) - \sigma_1 * w(t) * z(t) * l_2(t) + \sigma_2 * p_2(t), \\ u'(t) =& \alpha_2 - \delta_1 * u(t) - \sigma_1 * v(t) * u(t) * l_1(t) + \sigma_2 * p_1(t), \\ z'(t) =& \alpha_2 - \delta_1 * z(t) - \sigma_1 * w(t) * z(t) * l_2(t) + \sigma_2 * p_2(t), \\ k'(t) =& \alpha_1- \delta_1 * k(t) - \tau * k(t) * p_1(t) - \tau * k(t) * p_2(t), \\ k_1'(t) =& -\delta_1 * k_1(t) - \beta_1 * b(t) * k_1(t) + \gamma_2 * k_{12}(t) + \\ & \gamma_2 * k_{123}(t) + \beta_2 * k_1b(t) - \gamma_1 * k_1(t) * k_2(t) - \gamma_1 * k_1(t) * k_{23}(t) \\ &+ \tau * k(t) * p_1(t) + \tau * k_1k_2(t) * p_1(t), \\ k_{12}'(t) =& -\delta_1 * k_{12}(t) - \gamma_2 * k_{12}(t) + \gamma_1 * k_1(t) * k_2(t), \\ k_{123}'(t) =& -\gamma_2 * k_{123}(t) + \gamma_1 * k_1(t) * k_{23}(t), \\ k_1b'(t) =& \beta_1 * b(t) * k_1(t) - \delta_1 * k_1b(t) - \beta_2 * k_1b(t), \\ k_1k_2'(t) =& -\tau * k_1k_2(t) * p_1(t) + \tau * k(t) * p_2(t), \\ k_2'(t) =& \gamma_2 * k_{12}(t) - \delta_1 * k_2(t) - \gamma_1 * k_1(t) * k_2(t) + \tau * k_1k_2(t) * p_1(t) + \tau * k_{23}(t) * p_2(t), \\ k_{23}'(t) =& \gamma_2 * k_{123}(t) - \delta_1 * k_{23}(t) - \gamma_1 * k_1(t) * k_{23}(t) + \tau * k(t) * p_1(t) - \tau * k_{23}(t) * p_2(t), \\ k_3'(t) =& -\delta_1 * k_3(t) + \tau * k(t) * p_2(t) + \tau * k_{23}(t) * p_2(t), \\ p_1'(t) =& \sigma_1 * v(t) * u(t) * l_1(t) - \sigma_2 * p_1(t), \\ p_2'(t) =& \sigma_1 * w(t) * z(t) * l_2(t) - \sigma_2 * p_2(t) \end{align}Mathematical nonimplication f4
\begin{align} v'(t) =& \alpha_2 - \delta_1 * v(t) - \sigma_1 * v(t) * u(t) * l_1(t) + \sigma_2 * p_1(t), \\ w'(t) =& \alpha_2 - \delta_1 * w(t) - \sigma_1 * w(t) * z(t) * l_2(t) + \sigma_2 * p_2(t), \\ u'(t) =& \alpha_2 - \delta_1 * u(t) - \sigma_1 * v(t) * u(t) * l_1(t) + \sigma_2 * p_1(t), \\ z'(t) =& \alpha_2 - \delta_1 * z(t) - \sigma_1 * w(t) * z(t) * l_2(t) + \sigma_2 * p_2(t), \\ d'(t) =& \alpha_1- \delta_1 * d(t) - \beta_1 * d(t) * g_1(t) + \beta_2 * g_1d(t) - \tau * d(t) * p_2(t), \\ d_1'(t) =& -\delta_1 * d_1(t) - \gamma_1 * d_1(t) * g_1(t) + \gamma_2 * g_1d_1(t) + \tau * d(t) * p_2(t), \\ d_2'(t) =& -\delta_1 * d_2(t) + \tau * d(t) * p_2(t) + \tau * g_1d(t) * p_2(t), \\ g'(t) =& \alpha_1- \delta_1 * g(t) - \tau * g(t) * p_1(t), \\ g_1'(t) =& -\delta_1 * g_1(t) - \beta_1 * d(t) * g_1(t) - \gamma_1 * d_1(t) * g_1(t) \\ & + \beta_2 * g_1d(t) + \gamma_2 * g_1d_1(t) + \gamma_2 * g_1g_2(t) - \gamma_1 * g_1(t) * g_2(t), \\ g_1d'(t) =& \beta_1 * d(t) * g_1(t) - \delta_1 * g_1d(t) - \beta_2 * g_1d(t) - \tau * g_1d(t) * p_2(t), \\ g_1d_1'(t) =& \gamma_1 * d_1(t) * g_1(t) - \gamma_2 * g_1d_1(t) + \tau * g_1d(t) * p_2(t), \\ g_1g_2'(t) =& -\gamma_2 * g_1g_2(t) + \gamma_1 * g_1(t) * g_2(t) + \tau * g(t) * p_1(t), \\ g_2'(t) =& \gamma_2 * g_1g_2(t) - \delta_1 * g_2(t) - \gamma_1 * g_1(t) * g_2(t), \\ p_1'(t) =& \sigma_1 * v(t) * u(t) * l_1(t) - \sigma_2 * p_1(t), \\ p_2'(t) =& \sigma_1 * w(t) * z(t) * l_2(t) - \sigma_2 * p_2(t) \end{align}The function of light presence, denoted with $l(t)$, $l_1(t)$ or $l_2(t)$, is a piecewise function which equals 1 if the light is present and 0 otherwise. Functions $p_1$, $p_2$, $g$, $g_1$, $g_1d$, $g_1d_1$, $g_1g_2$, $g_1i$, $g_2$, $c$, $c_1$, $c_2$, $c_2d$, $cd$, $cd_2$, $w$, $z$, $d$, $d_1$, $d_2$, $k$, $k_1$, $k_{12}$, $k_{123}$, $k_1b$, $k_1k_2$, $k_2$, $k_{23}$, $k_3$, $i$, $b$, $k$, $v$, $u$, $w$, $z$ present concentrations of the equally labelled proteins. The constants used for the model are described in tab:refs .
Description | Name | Rate | Reference |
---|---|---|---|
protein production rate | $\alpha$ | 3.5 * 20$^{-2}$ nMs$^{-1}$ |
|
light inducible split protease production rate | $\alpha_2$ | 7 * 10$^{-1}$ nMs$^{-1}$ | protein:protease DNA ratio is 1:20 |
protein degradation rate | $\delta_1$ | Log[2] / (3600 * 9) $s^{-1}$ |
|
light inducible split protease dissociation rate | $\sigma_2$ | Log[2] / (60 * 5.5) s$^{-1}$ |
|
light inducible split protease association rate | $\sigma_1$ | 1 nM$^{-1}$ s$^{-1}$ |
|
protease cleavage rate | $\tau$ | 1.2 * 10$^-6$ nM$^-1$ s$^{-1}$ |
|
stronger coiled coils association rate | $\beta_1$ | 3.17 * 10$^{-3}$ nM$^{-1}$ s$^{-1}$ |
|
stronger coiled coils dissociation rate | $\beta_2$ | 2 * 10$^{-4}$ s$^{-1}$ |
|
weaker coiled coils association rate | $\gamma_1$ | 7.3 * 10$^{-6}$ nM$^{-1}$ s$^{-1}$ |
|
weaker coiled coils dissociation rate | $\gamma_2$ | 1.67 * 10$^{-1}$ s$^{-1}$ |
|
time of light exposure | / | 60 s | estimated from experimental results |
Results
We simulated the dynamics of established logic gates with the numerical integration of their mathematical models described in the previous paragraphs. The results of our simulations are shown in fig:buffer , fig:nor , fig:imply and fig:nimply . They confirm our assumption that all four types of logic functions offer short delay compared to their equivalents based on genetic regulatory networks. The rise and fall times of our gates are simulated to be at around 70 seconds compared to hours that transcription regulation circuits usually require.
![](https://static.igem.org/mediawiki/2016/7/7a/T--Slovenia--5.5.6.png)
![](https://static.igem.org/mediawiki/2016/2/27/T--Slovenia--5.5.7.png)
![](https://static.igem.org/mediawiki/2016/0/06/T--Slovenia--5.5.8.png)
![](https://static.igem.org/mediawiki/2016/1/11/T--Slovenia--5.5.9.png)
Our system also allows us to shorten the lifetime of the output signal without significantly reducing its concentrations, by adding degradation tags to the output protein. The high output times achieved can even be similar to the input light induction time of 1 minute. These two characteristics can importantly influence several sequential induction of logic gates and the further development of several layered logic circuits.
![](https://static.igem.org/mediawiki/2016/0/08/T--Slovenia--5.5.10.png)