Difference between revisions of "Team:Slovenia/CoiledCoilInteraction"

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                 <div class="main ui citing justified container">
 
                 <div class="main ui citing justified container">
 
                     <h2>Coiled Coil interaction model</h2>
 
                     <h2>Coiled Coil interaction model</h2>
−
                     <p>Logic operations in biological systems have been tested with several approaches
+
                     <div class="ui segment">
−
                        <x-ref>Singh2014</x-ref>
+
                        <p>Logic operations in biological systems have been tested with several approaches
−
                        . Our project
+
                            <x-ref>Singh2014</x-ref>
−
                        relies on the reconstitution of split protein promoted by coiled coil (CC) dimerization. The
+
                            . Our project
−
                        interaction between CC peptides can be finely tuned
+
                            relies on the reconstitution of split protein promoted by coiled coil (CC) dimerization. The
−
                        <x-ref>Woolfson2005, Gradisar2011, Negron2014</x-ref>
+
                            interaction between CC peptides can be finely tuned
−
                        , thereby CCs offers a flexible and
+
                            <x-ref>Woolfson2005, Gradisar2011, Negron2014</x-ref>
−
                        versatile platform in terms of designing logic operation in vivo. With the purpose of
+
                            , thereby CCs offers a flexible and
−
                        understanding the relation that underlies the interaction between coiled coil peptides and
+
                            versatile platform in terms of designing logic operation in vivo. With the purpose of
−
                        therefore using them in logic gates, we designed the following model (
+
                            understanding the relation that underlies the interaction between coiled coil peptides and
−
                        <ref>5.4.1.</ref>
+
                            therefore using them in logic gates, we designed the following model (
−
                        ). Our system is based on constructs that have been characterized in mammalian cells in the
+
                            <ref>5.4.1.</ref>
−
                        context of <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic">logic function
+
                            ). Our system is based on constructs that have been characterized in mammalian cells in the
−
                            design</a>. Two orthogonal CC segment, A and b, fused together in on chain can bind each
+
                            context of <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic">logic
−
                        other and form a stable CC pair. This complex exists in combination with the peptide B, which
+
                                function
−
                        can also bind the peptide A and has a different affinity from the peptide b. The linker that
+
                                design</a>. Two orthogonal CC segment, A and b, fused together in on chain can bind each
−
                        connects A and b can be cleaved by a generic protease (e.g. TEV), this irreversible reaction
+
                            other and form a stable CC pair. This complex exists in combination with the peptide B,
−
                        shift the equilibrium towards a state in which all of the three peptides are free in solution
+
                            which
−
                        and therefore compete for binding. In our experiments, a similar system as the generic coils A
+
                            can also bind the peptide A and has a different affinity from the peptide b. The linker that
−
                        and B was fused to the <a
+
                            connects A and b can be cleaved by a generic protease (e.g. TEV), this irreversible reaction
−
                                href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters">split reporter
+
                            shift the equilibrium towards a state in which all of the three peptides are free in
−
                            firefly luciferase</a>.
+
                            solution
−
                    </p>
+
                            and therefore compete for binding. In our experiments, a similar system as the generic coils
 +
                            A
 +
                            and B was fused to the <a
 +
                                    href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters">split
 +
                                reporter
 +
                                firefly luciferase</a>.
 +
                        </p>
  
−
                    <div style="float:left; width:100%">
+
                        <div style="float:left; width:100%">
−
                        <figure data-ref="5.4.1.">
+
                            <figure data-ref="5.4.1.">
−
                            <img
+
                                <img
−
                                src="https://static.igem.org/mediawiki/2016/9/98/T--Slovenia--5.4.1.png">
+
                                        src="https://static.igem.org/mediawiki/2016/9/98/T--Slovenia--5.4.1.png">
−
                            <figcaption><b> Scheme representing the CC interaction model </b><br/> The two state system
+
                                <figcaption><b> Scheme representing the CC interaction model </b><br/> The two state
−
                                is considered at inducible by activity of TEV protease and signal both before and after
+
                                    system
−
                                cleavage is represented as reconstitution on split firefly luciferase reporter.
+
                                    is considered at inducible by activity of TEV protease and signal both before and
−
                            </figcaption>
+
                                    after
−
                        </figure>
+
                                    cleavage is represented as reconstitution on split firefly luciferase reporter.
−
                    </div>
+
                                </figcaption>
−
                    <p>The relationship between the signal before and after cleavage by proteases is represented by the
+
                            </figure>
−
                        difference [AB] - [AB-b]. In order to understand the optimal combination of dissociation
+
                        </div>
−
                        constant required to obtain a good signal we solved two systems of equations set up considering
+
                        <p>The relationship between the signal before and after cleavage by proteases is represented by
−
                        the two state of the reaction scheme (“Before cleavage and “After cleavage”) as separate phases
+
                            the
−
                        of the reaction and additionally, considering cleavage as an irreversible and complete
+
                            difference [AB] - [AB-b]. In order to understand the optimal combination of dissociation
−
                        reaction.</p>
+
                            constant required to obtain a good signal we solved two systems of equations set up
−
                    <p>Given values for total concentrations and Kd (from 10<sup>-9</sup> to 10<sup>-3</sup> M) the
+
                            considering
−
                        equations, for the
+
                            the two state of the reaction scheme (“Before cleavage and “After cleavage”) as separate
−
                        reaction constants \eqref{1.1-2} - \eqref{2.1-2} and for mass conservation \eqref{1.3-4} -
+
                            phases
−
                        \eqref{2.3-5}, were solved for the
+
                            of the reaction and additionally, considering cleavage as an irreversible and complete
−
                        species at equilibrium.</p>
+
                            reaction.</p>
−
                    Before cleavage
+
                        <p>Given values for total concentrations and Kd (from 10<sup>-9</sup> to 10<sup>-3</sup> M) the
−
                    \begin{equation}
+
                            equations, for the
−
                    \ce{Axb + B <=>[Kd_x] A-b + B <=>[Kd_B] AB-b}  
+
                            reaction constants \eqref{1.1-2} - \eqref{2.1-2} and for mass conservation \eqref{1.3-4} -
−
                    \end{equation}
+
                            \eqref{2.3-5}, were solved for the
−
                    \begin{align}
+
                            species at equilibrium.</p>
−
                    Kd_x &= \frac{[A-b]}{[Axb]} \label{1.1-2}\\
+
                        Before cleavage
−
                    Kd_B &= \frac{[A-b] * [B]}{[AB - b]} \\
+
                        \begin{equation}
−
                    c_B &= [B] + [AB-b]\\
+
                        \ce{Axb + B <=>[Kd_x] A-b + B <=>[Kd_B] AB-b}
−
                    c_A-b &= [A-b]+[Axb]+[AB-b] \label{2.1-2}
+
                        \end{equation}
−
                    \end{align}
+
                        \begin{align}
−
                    After cleavage
+
                        Kd_x &= \frac{[A-b]}{[Axb]} \label{1.1-2}\\
−
                    \begin{equation}
+
                        Kd_B &= \frac{[A-b] * [B]}{[AB - b]} \\
−
                    \ce{Ab + B <=>[Kd_b] A + b + B <=>[Kd_B] AB + b}  
+
                        c_B &= [B] + [AB-b]\\
−
                    \end{equation}
+
                        c_A-b &= [A-b]+[Axb]+[AB-b] \label{2.1-2}
−
                    \begin{align}
+
                        \end{align}
−
                    Kd_b &= \frac{[A] * [b]}{[Ab]} \label{1.3-4}\\
+
                        After cleavage
−
                    Kd_B &= \frac{[A] * [B]}{[AB]} \\
+
                        \begin{equation}
−
                    c_A &= [A]+[AB]+[Ab]\\
+
                        \ce{Ab + B <=>[Kd_b] A + b + B <=>[Kd_B] AB + b}
−
                    c_B &= [B] +[AB]\\
+
                        \end{equation}
−
                    c_b &= [b] + [Ab] \label{2.3-5}
+
                        \begin{align}
−
                    \end{align}
+
                        Kd_b &= \frac{[A] * [b]}{[Ab]} \label{1.3-4}\\
 +
                        Kd_B &= \frac{[A] * [B]}{[AB]} \\
 +
                        c_A &= [A]+[AB]+[Ab]\\
 +
                        c_B &= [B] +[AB]\\
 +
                        c_b &= [b] + [Ab] \label{2.3-5}
 +
                        \end{align}
  
−
                    >external text
+
                        >external text
−
                    The two systems are connected by the relation between the dissociation constants $Kd_b$ and $Kd_x$,
+
                        The two systems are connected by the relation between the dissociation constants $Kd_b$ and
−
                    \begin{equation}
+
                        $Kd_x$,
−
                    Kd_x = Kd_b * 4 * 10^{-3} M^{-1}
+
                        \begin{equation}
−
                    \end{equation}
+
                        Kd_x = Kd_b * 4 * 10^{-3} M^{-1}
−
                    This relation approximates the higher affinity between the coils A and b when they are covalently
+
                        \end{equation}
−
                    linked by a short peptide (as in the system “Before cleavage”)
+
                        This relation approximates the higher affinity between the coils A and b when they are
−
                    <x-ref>Moran1999, Zhou2004</x-ref>
+
                        covalently
−
                    .
+
                        linked by a short peptide (as in the system “Before cleavage”)
−
                    <p>The results have been plotted varying the Kd for the interaction of A with both B and b, against
+
                        <x-ref>Moran1999, Zhou2004</x-ref>
−
                        the difference [AB] - [AB-b], where [AB] is considered the signal after cleavage and [AB-b] the
+
                        .
−
                        signal before cleavage (leakage). The system revealed that in order to obtain a high difference
+
                        <p>The results have been plotted varying the Kd for the interaction of A with both B and b,
−
                        between signal and leakage a high affinity of the coil B for the coil A (low $Kd_B$) is
+
                            against
−
                        required,
+
                            the difference [AB] - [AB-b], where [AB] is considered the signal after cleavage and [AB-b]
−
                        while on the other hand an excessive destabilization of the autoinhibitory coil b (high $Kd_b$)
+
                            the
−
                        would prevent the signal to be visible (
+
                            signal before cleavage (leakage). The system revealed that in order to obtain a high
−
                        <ref>5.4.2.</ref>
+
                            difference
−
                        ).
+
                            between signal and leakage a high affinity of the coil B for the coil A (low $Kd_B$) is
−
                    </p>
+
                            required,
−
                    <div style="float:left; width:100%">
+
                            while on the other hand an excessive destabilization of the autoinhibitory coil b (high
−
                        <figure data-ref="5.4.2.">
+
                            $Kd_b$)
−
                            <img
+
                            would prevent the signal to be visible (
−
                                src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--5.4.2.png">
+
                            <ref>5.4.2.</ref>
−
                            <figcaption><b> Difference between [AB] and [AB-b] depending on the ratio of Kd
+
                            ).
−
                                values.</b><br/> The plots display the difference (M) between the signal before after
+
                        </p>
−
                                and the proteolytic cleavage (left) and the concentration of the species responsible for
+
                        <div style="float:left; width:100%">
−
                                leakage [AB-b] (right) in a range of different Kd values.
+
                            <figure data-ref="5.4.2.">
−
                            </figcaption>
+
                                <img
−
                        </figure>
+
                                        src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--5.4.2.png">
 +
                                <figcaption><b> Difference between [AB] and [AB-b] depending on the ratio of Kd
 +
                                    values.</b><br/> The plots display the difference (M) between the signal before
 +
                                    after
 +
                                    and the proteolytic cleavage (left) and the concentration of the species responsible
 +
                                    for
 +
                                    leakage [AB-b] (right) in a range of different Kd values.
 +
                                </figcaption>
 +
                            </figure>
 +
                        </div>
 +
 
 +
                        <p>This relationship suggested to try using a different version of the coiled coils available in
 +
                            the
 +
                            toolset already used by the <a href="https://2009.igem.org/Team:Slovenia">Slovenian iGEM 2009
 +
                                team</a>
 +
                            <x-ref>Gradisar2011</x-ref>
 +
                            .In order to
 +
                            obtain a detectable signal for <a
 +
                                    href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic">logic operation
 +
                                in
 +
                                vivo </a> we decided
 +
                            to use an inhibitory coiled coil, which would be displaced by the second coiled coil with
 +
                            higher
 +
                            affinity, only once is cleaved off its partner ($ Kd_B \gt Kd_b $). In doing so we selected
 +
                            P3 as
 +
                            B and
 +
                            P3mS as b, these two coiled coil peptides present only few substitutions and the higher
 +
                            solubility of P3mS (b), which presents Gln and Ser instead of Ala in b and c position of the
 +
                            heptads, would favour the dissociation. We also tried differently destabilized versions of
 +
                            P3
 +
                            and it turned out that, as in the forehead described model, an excessive destabilization
 +
                            (obtained by substituting a and d positions with Ala) leads to a small difference of the
 +
                            signal
 +
                            before and after cleavage. Using a slightly destabilized coiled coil (P3mS-2A), which
 +
                            presents
 +
                            only 2 alanines in the second heptad, the signal after cleavage reached its maximum of 16
 +
                            folds.
 +
                            (MISSING Link to Figure 4.12.9.)
 +
                        </p>
 
                     </div>
 
                     </div>
−
 
−
                    <p>This relationship suggested to try using a different version of the coiled coils available in the
 
−
                        toolset already used by the <a href="https://2009.igem.org/Team:Slovenia">Slovenian iGEM 2009
 
−
                            team</a>
 
−
                        <x-ref>Gradisar2011</x-ref>
 
−
                        .In order to
 
−
                        obtain a detectable signal for <a
 
−
                                href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic">logic operation in
 
−
                            vivo </a> we decided
 
−
                                to use an inhibitory coiled coil, which would be displaced by the second coiled coil with higher
 
−
                                affinity, only once is cleaved off its partner ($ Kd_B \gt Kd_b $). In doing so we selected
 
−
                        P3 as
 
−
                        B and
 
−
                        P3mS as b, these two coiled coil peptides present only few substitutions and the higher
 
−
                        solubility of P3mS (b), which presents Gln and Ser instead of Ala in b and c position of the
 
−
                        heptads, would favour the dissociation. We also tried differently destabilized versions of P3
 
−
                        and it turned out that, as in the forehead described model, an excessive destabilization
 
−
                        (obtained by substituting a and d positions with Ala) leads to a small difference of the signal
 
−
                        before and after cleavage. Using a slightly destabilized coiled coil (P3mS-2A), which presents
 
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                        only 2 alanines in the second heptad, the signal after cleavage reached its maximum of 16 folds.
 
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                        (MISSING Link to Figure 4.12.9.)
 
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Revision as of 18:38, 17 October 2016

Model Logic

Project Achievements Medal requirements Idea

Coiled Coil interaction model

Logic operations in biological systems have been tested with several approaches Singh2014 . Our project relies on the reconstitution of split protein promoted by coiled coil (CC) dimerization. The interaction between CC peptides can be finely tuned Woolfson2005, Gradisar2011, Negron2014 , thereby CCs offers a flexible and versatile platform in terms of designing logic operation in vivo. With the purpose of understanding the relation that underlies the interaction between coiled coil peptides and therefore using them in logic gates, we designed the following model ( 5.4.1. ). Our system is based on constructs that have been characterized in mammalian cells in the context of logic function design. Two orthogonal CC segment, A and b, fused together in on chain can bind each other and form a stable CC pair. This complex exists in combination with the peptide B, which can also bind the peptide A and has a different affinity from the peptide b. The linker that connects A and b can be cleaved by a generic protease (e.g. TEV), this irreversible reaction shift the equilibrium towards a state in which all of the three peptides are free in solution and therefore compete for binding. In our experiments, a similar system as the generic coils A and B was fused to the split reporter firefly luciferase.

Scheme representing the CC interaction model
The two state system is considered at inducible by activity of TEV protease and signal both before and after cleavage is represented as reconstitution on split firefly luciferase reporter.

The relationship between the signal before and after cleavage by proteases is represented by the difference [AB] - [AB-b]. In order to understand the optimal combination of dissociation constant required to obtain a good signal we solved two systems of equations set up considering the two state of the reaction scheme (“Before cleavage and “After cleavage”) as separate phases of the reaction and additionally, considering cleavage as an irreversible and complete reaction.

Given values for total concentrations and Kd (from 10-9 to 10-3 M) the equations, for the reaction constants \eqref{1.1-2} - \eqref{2.1-2} and for mass conservation \eqref{1.3-4} - \eqref{2.3-5}, were solved for the species at equilibrium.

Before cleavage \begin{equation} \ce{Axb + B <=>[Kd_x] A-b + B <=>[Kd_B] AB-b} \end{equation} \begin{align} Kd_x &= \frac{[A-b]}{[Axb]} \label{1.1-2}\\ Kd_B &= \frac{[A-b] * [B]}{[AB - b]} \\ c_B &= [B] + [AB-b]\\ c_A-b &= [A-b]+[Axb]+[AB-b] \label{2.1-2} \end{align} After cleavage \begin{equation} \ce{Ab + B <=>[Kd_b] A + b + B <=>[Kd_B] AB + b} \end{equation} \begin{align} Kd_b &= \frac{[A] * [b]}{[Ab]} \label{1.3-4}\\ Kd_B &= \frac{[A] * [B]}{[AB]} \\ c_A &= [A]+[AB]+[Ab]\\ c_B &= [B] +[AB]\\ c_b &= [b] + [Ab] \label{2.3-5} \end{align} >external text The two systems are connected by the relation between the dissociation constants $Kd_b$ and $Kd_x$, \begin{equation} Kd_x = Kd_b * 4 * 10^{-3} M^{-1} \end{equation} This relation approximates the higher affinity between the coils A and b when they are covalently linked by a short peptide (as in the system “Before cleavage”) Moran1999, Zhou2004 .

The results have been plotted varying the Kd for the interaction of A with both B and b, against the difference [AB] - [AB-b], where [AB] is considered the signal after cleavage and [AB-b] the signal before cleavage (leakage). The system revealed that in order to obtain a high difference between signal and leakage a high affinity of the coil B for the coil A (low $Kd_B$) is required, while on the other hand an excessive destabilization of the autoinhibitory coil b (high $Kd_b$) would prevent the signal to be visible ( 5.4.2. ).

Difference between [AB] and [AB-b] depending on the ratio of Kd values.
The plots display the difference (M) between the signal before after and the proteolytic cleavage (left) and the concentration of the species responsible for leakage [AB-b] (right) in a range of different Kd values.

This relationship suggested to try using a different version of the coiled coils available in the toolset already used by the Slovenian iGEM 2009 team Gradisar2011 .In order to obtain a detectable signal for logic operation in vivo we decided to use an inhibitory coiled coil, which would be displaced by the second coiled coil with higher affinity, only once is cleaved off its partner ($ Kd_B \gt Kd_b $). In doing so we selected P3 as B and P3mS as b, these two coiled coil peptides present only few substitutions and the higher solubility of P3mS (b), which presents Gln and Ser instead of Ala in b and c position of the heptads, would favour the dissociation. We also tried differently destabilized versions of P3 and it turned out that, as in the forehead described model, an excessive destabilization (obtained by substituting a and d positions with Ala) leads to a small difference of the signal before and after cleavage. Using a slightly destabilized coiled coil (P3mS-2A), which presents only 2 alanines in the second heptad, the signal after cleavage reached its maximum of 16 folds. (MISSING Link to Figure 4.12.9.)

References