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2024-03-28T20:42:00Z
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http://2012.igem.org/Team:Slovenia/ModelingDerivation
Team:Slovenia/ModelingDerivation
2013-06-03T18:02:25Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
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<h1>Deterministic model derivation - genetic switch</h1><br />
<p><br />
The deterministic model for the mutual repressor switch was derived in the following way.<br />
</p><p><br />
We assumed the following binding site states were possible:<br />
<p><img src="https://static.igem.org/mediawiki/2012/2/2c/Svn12_model_derivation1.PNG"/></p><br />
</p><br />
<br />
<p>Active promoter state is a state leading to gene expression.<br />
</p><br />
<br />
<p>Construct 1 promoter state transitioning is described by a reaction:<img src="https://static.igem.org/mediawiki/2012/f/ff/Svn12_derivation1_eq1.png"/><br />
</p><br />
<!--<br />
<p>Assuming equilibrium of binding and unbinding, we can write:<br />
<img src="https://static.igem.org/mediawiki/2012/f/f7/Svn12_derivation1_eq2.png"/><br />
</p>--><br />
<br />
<p><br />
The fractional occupancy of construct 1 promoter, f<sub>1</sub>, can be expressed as a ratio of active states to all states:<br />
<img src="https://static.igem.org/mediawiki/2012/b/b7/Svn12_derivation1_eq3.png" /><br />
</p><br />
<br />
<p><br />
The equations for other constructs take the same form.<br />
</p><br />
<br />
<p><br />
Assuming equilibrium of binding and unbinding, it follows that the fractional occupancy for construct 1 promoter is:<br />
<img src="https://static.igem.org/mediawiki/2012/b/ba/Svn12_derivation1_eq4.png" /><br />
</p><br />
<br />
<p>To account for non-linearity, an exponent n<sub>1</sub> was added, and the equation generalized to:<br />
<img src="https://static.igem.org/mediawiki/2012/a/a9/Svn12_derivation1_eq5.png" /><br />
</p><br />
<br />
<br />
<p><br />
K<sub>r</sub> is the amount of TAL-A:KRAB it takes for f<sub>1</sub> to be equal to 50% when n<sub>1</sub>=k<sub>1</sub>=1.<br />
</p><br />
<br />
<p><br />
Derivation for constructs 2, 3 and 4 was similar except for different transcription factor names.<br />
</p><br />
<br />
<p><br />
Fractional occupancies were then used to construct a set of ordinary differential equations representing each protein production. Because each protein can be produced from different constructs, production rates (including leaky rates) were summed together. E.g., because TAL-B:KRAB is produced from both constructs 1 and 3, fractional occupancies f<sub>1</sub> and f<sub>3</sub> were used and corresponding terms summed to obtain:<br/><br/><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Svn12_mrs_det_d3_talbkrab.png" /><br />
</p><br />
<br />
<p><br />
Since construct 5 promoter has no binding sites and is active at all times, fractional occupancy of the promoter is equal to 1.<br />
</p><br />
<br />
<br />
<p><br />
The positive feedback loop switch model was derived in a similar manner.<br />
</p><br />
<br />
<br />
<br />
<br />
<hr><br />
<b><br />
<a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'>Deterministic model of the mutual repressor switch</a><br/><br />
<a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'>Deterministic model of the positive feedback loop switch</a><br />
<br />
</b><br />
<br />
<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ModelingMethods
Team:Slovenia/ModelingMethods
2013-03-19T15:43:57Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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</a><br />
<br />
<h1>Modeling methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#intro">Introduction</a><br/></li><br />
<li><a href="#cooperativity">Cooperativity</a></li><br />
<li><a href="#deterministic">Deterministic modeling</a></li><br />
<li><a href="#stochastic">Stochastic modeling</a></li><br />
<li><a href="#quantitative">Quantitative model and stability analysis</a></li><br />
<li><a href="#csim">C#Sim - algorithmic modeling</a></li><br />
<li><a href="#source">Source code</a></li><br />
</ul><br />
</p><br />
<br />
<br />
<br />
<br />
<h2><a name="intro">Introduction</a></h2><br />
<p><br />
In order to examine (i.e. simulate) the proposed genetic switches <i>in silico</i>, different modeling approaches were used. First, a deterministic model based on the probabilistic interpretation of gene regulation was constructed for each type of a genetic switch. Next, a stochastic simulation was performed to take inherent stochastic dynamics of gene expression into account. To further verify the results obtained using these methods, we also developed a quantitative model that builds upon experimental data. Moreover, we developed a modeling algorithm to more explicitly simulate transcription factor binding, considering number of available binding sites and competitive binding. Each modeling approach is discussed in the following sections. We also discuss the notion of cooperativity in the context of bistability.<br />
</p><br />
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<br />
<br />
<br />
<h2><a name="cooperativity">Cooperativity</a></h2><br />
<p><br />
<p><br />
It is often assumed that functional cooperativity (e.g. multimeric regulation) is required for bistability. However, it has been shown theoretically that bistability can emerge in systems without multimeric regulation, provided that at least one regulatory autoloop is present. (Widder et al., 2009). Furthermore, <i>in silico</i> analysis has shown the existence of bistable architectures without the transcription factor cooperativity typically associated with switch-like properties (Siegal-Gaskins et al., 2011). An essential feature of these proposed architectures was the competitive binding of two transcription factors to the promoter.<br />
</p><br />
<p><br />
In terms of modeling, sigmoidal functions (characterizing the rate of change dP/dt) – arising from (Hill) exponents greater than one - are often equated with molecular cooperativity (the way the transcription factor binds to a promoter). However, as non-linearity and multi-stability can arise without assuming molecular cooperativity, it has been suggested that this is not an accurate proposition and that mathematical or functional cooperativity – referring to a sigmoidal function arising from system equations - should not automatically be interpreted as molecular cooperativity (Andrecut et al., 2011). One reason for this is that model equations represent a significant simplification of actual biological dynamics of gene expression, which include a large number of reactions not explicitly considered in modeling, such as reactions describing chromosome opening and transcription initiation.<br />
</p><br />
<br />
<p><br />
For this reasons, we believe that sigmoidal behavior alone – arising in some of our models for transcription factors’ exponent values (non-linearity) greater than 1 - should not by default be interpreted as molecular cooperativity. Thus, in the context of modeling, with the term cooperativity we mean functional cooperativity greater than 1. Functional cooperativity equal to 1 is referred to as no cooperativity.<br />
</p><br />
<br />
<p><br />
Indeed, in case of our positive feedback loop switch – which contains both competitive binding and regulatory autoloops - even deterministic models predict experimentally-verified bistability at low (i.e. close to 1; deterministic fractional occupancy model) or no (quantitative model) functional cooperativity. Our stochastic model of the positive feedback loop switch also predicts bistability is possible without cooperativity.<br />
</p><br />
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</p><br />
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<br />
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<br />
<h2><a name="deterministic">Deterministic modeling</a></h2><br />
<p><br />
<p><br />
Our deterministic models are based on fractional occupancy, a quantity which expresses the degree of saturation at the transcription factor binding site (Sauro, 2012). Fractional occupancy can be expressed as a ratio of active binding site states – i.e. states leading to gene expression – to all possible binding site states:<p><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Svn12_modeling_eqn0.png"/></p><br />
<br/><br />
As such, it can be interpreted as a probability of a promoter being active, or a probability that transcription and/or translation will occur. Gene expression in our model, in turn, is proportional to this probability.<br />
</p><br />
<p><br />
Two main possibilities are distinguished in our models depending on the type of the promoter. In case of a minimal promoter, binding of a transcriptional activator (leading to an active state) is required for transcription initiation. Other states – binding site being free or bound by a repressor - are considered inactive. In case of a constitutive promoter, binding of a transcriptional repressor leads to an inactive state, while unbound (or activator-bound) states are active, hence leading to gene expression.<br />
</p><br />
<p><br />
We assumed large species concentrations compared to the number of available binding sites. We also assumed fast transitions between promoter states, i.e. transcription factor binding and unbinding occurs much faster than transcription/translation, which in this type of deterministic models are represented as a single step of protein production. Another simplification made was a representation of multiple repeats of a binding site as a single binding site (e.g. a specific TAL-A:KRAB binding site may in reality have 10 repeats, while in our models it is represented as a single site). As the purpose of our deterministic models is to provide an approximate, basic characterization of the proposed logic, we consider these simplifications acceptable. Other models, such as C#Sim models (described later), try to formalize some of these aspects in more detail.<br />
</p><br />
<br />
<p><br />
The mathematical framework which describes protein production and into which we incorporate fractional occupancy is represented as a set of ordinary differential equations (ODEs) of the form:<br />
<p><img src="https://static.igem.org/mediawiki/2012/1/16/Svn12_modeling_eqn1.png"/></p><br />
and is roughly based on a framework proposed in (Kaern et al., 2005). Here:<br />
<ul style="margin-left:15px;"><br />
<li>[Protein] is protein concentration at a given time;</li><br />
<li>k is a constant specifying protein production rate in case of an active promoter.</li><br />
<li>kb is a constant specifying the amount of leaky gene expression, meaning protein production that takes place even in the case of inactive (i.e. repressed constitutive or non-activated minimal) promoter.</li><br />
<li>dg is a constant specifying protein degradation rate.</li><br />
<li>P(active) is the probability of a promoter being in an active state – this probability is equal to fractional occupancy f.</li><br />
<li>P(inactive) is the probability of a promoter being inactive and is equal to (1-f).</li><br />
</ul><br />
</p><br />
<br />
<p><br />
No specific quantitative data describing e.g. transcription, translation or degradation rates of TAL effectors was available to us. For this reason, the parameter values (specifying e.g production or degradation rates) in our models were assumed based on commonly accepted propositions, such as that production (i.e. transcription and translation) rates of proteins are (usually) higher than their degradation rates. Parameter values that were used in our simulations should hence only be considered in relative terms (e.g. as protein production to degradation ratios). We also assumed parameter symmetry of different TAL effectors used (TAL-A and TAL-B), because we did not expect that one would have e.g. a production rate significantly higher than the other.<br />
</p><br />
<br />
<p>All deterministic models were implemented in MATLAB.</p><br />
</p><br />
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<br />
<h2><a name="stochastic">Stochastic modeling</a></h2><br />
<p><br />
<p><br />
Gene expression is an inherently stochastic process and deterministic simulation is often not satisfactory. The required conditions for the two approaches to be similar are large system size (high mRNA and protein numbers) and fast promoter kinetics (Kaern et al., 2005), but these conditions are not always met. Furthermore, stochastic models can capture the individuality of single cells, as opposed to deterministic models that can only predict the average behavior of a cell population (Kaern et al., 2003). For this reason we performed stochastic simulations of the proposed genetic switches. Our results were obtained using SGN Sim, a stochastic genetic networks simulator (Ribeiro et al., 2007) based on stochastic simulation algorithm (SSA).<br />
</p><br />
<br />
<p><br />
Competitive binding between an activator and a repressor exists in our positive feedback loop switch. This can lead to three major minimal promoter states: the promoter being free (i. e. unbound - neither an activator nor a repressor is bound, zero activation is present), bound by a repressor or bound by an activator. When minimal promoter is unbound, only leaky expression occurs. If bound by an activator, this causes high activated expression levels. Bound repressor causes the expression levels to drop even below the ones achieved for the unbound (non-activated) state.<br />
</p><br />
<br />
<p><br />
Our deterministic models made no clear distinction between free (unbound) minimal promoter state and a repressor-bound state – in both cases inactive promoter state was assumed, leading to only leaky expression. In our stochastic models we improved this by introducing different expression levels for all three states, achieving a more realistic formalization.<br />
</p><br />
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</p><br />
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<br />
<br />
<br />
<br />
<br />
<h2><a name="quantitative">Quantitative model and stability analysis</a></h2><br />
<p><br />
Based on experimental data we constructed what we refer to as a quantitative model. Please see <a href="https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel">Quantitative and stability model</a> for details. To get a better idea of the functioning of the model, an <a href="https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations">interactive web application</a> was developed.</p><br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h2><a name="csim">C#Sim - algorithmic modeling</a></h2><br />
<p><br />
<p>We developed a new modeling algorithm called C#Sim. The main purpose of this was:<br />
<ul style="margin-left:30px;"><br />
<li>to explicitly model a <b>limited number of binding site repeats;</b></li><br />
<li>to explicitly model <b>competitive binding</b> of activators and repressors;</li><br />
<li>to construct a <b>new modeling approach</b> that incorporates stochasticity of gene expression into an otherwise deterministic approach.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
The assumptions made here were similar to that of deterministic modeling. First, we assumed that high concentrations of mRNA and proteins existed compared to the number of available binding sites that transcription factors could bind to. Second, we assumed that binding of transcription factors to binding sites was much faster than transcription and translation.<br />
</p><br />
<br />
<br />
<p><br />
A feature of C#Sim is that it operates with <b>discrete numbers</b> – at each step of the simulation, mRNA and protein amounts are non-negative integers. Each mRNA and protein molecule is represented as an <b>independent entity</b> – or object - defined by its parameters and interactions. An <b>object-oriented programming approach</b> was used to achieve this. At the moment, C#Sim supports the following entity types, implemented as classes:<br />
<br />
<!-- table --><br />
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<th>mRNA</th><br />
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<tr class="normal"><br />
<td class="normal"><br />
<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>birth time (integer) – tells at what simulation step this mRNA entity was created (transcribed); used for modeling transcription-translation delay;</li><br />
<li><br />
alive (true/false) – tells if this mRNA is alive and subject to translation (true) or not.</li><br />
</ul><br />
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</td><br />
</tr> <br />
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<th>Protein</th><br />
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<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>birth time (integer) – tells at what simulation step this protein entity was created (translated);</li><br />
<li>alive (true/false) – tells if this protein is alive (true), e.g. it can function as a transcription factor.</li><br />
</ul><br />
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</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<!-- /table --><br />
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<th>Binding site</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><br />
<td class="normal"><br />
<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>capacity (integer) – the number of binding site repeats; each repeat can be bound by one protein entity;</li><br />
<li>amount of bound activator (integer) – the number of activator entities bound to this binding site;</li><br />
<li>amount of bound repressor (integer) – the number of repressor entities bound to this binding site.</li><br />
</ul><br />
<br />
</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<!-- /table --><br />
<br />
<!-- table --><br />
<table class="normal"><br />
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<th>Promoter</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><br />
<td class="normal"><br />
<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>promoter type – either minimal or constitutive;</li><br />
<li>list of promoter binding sites;</li><br />
<li>active mRNA transcription rate, which determines the number of mRNA entities to produce at each simulation step for activated promoter (in case of a minimal promoter) or non-repressed promoter (in case of a constitutive promoter);</li><br />
<li>inactive (leaky) mRNA transcription rate, which determines the number of mRNA entities that can be produced even when the promoter is not activated (for minimal promoter), or when the promoter is repressed (for constitutive promoter);</li><br />
<li>the amount of all repressors and activators bound to all promoter binding sites.</li><br />
</ul><br />
<br />
</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<!-- /table --><br />
<br />
<br />
<br />
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<table class="normal"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Gene</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><br />
<td class="normal"><br />
<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>gene's promoter;</li><br />
<li><br />
translation rate, describing the number of proteins produced per each transcribed alive mRNA entity at each simulation step;</li><br />
<li>mRNA and protein degradation rate (i.e. the percentage of mRNA and protein degraded at each simulation step).</li><br />
</ul><br />
<br />
</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<!-- /table --><br />
</p><br />
<br />
<p><br />
Note that the majority of these parameters are computed automatically, not specified by the user (programmer). The parameters specified by the user (when defining the network structure) are: binding sites' capacities, promoter types, list of promoter binding sites, active and inactive transcription rates, a promoter for each gene, translation rates and degradation rates. A delay between transcription and translation can also be specified.<br />
</p><br />
<br />
<p><br />
Using these entities, a hierarchy of gene regulatory network can quickly be constructed. Furthermore, the approach is highly modular and extendible with new dynamics. <!--For examples, please take a look at the <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a>, which contains both our switches implemented in C# language.--><br />
</p><br />
<br />
<p><br />
Other parameters also define the behavior of the mentioned entity types – they are listed and explained in more detail in the algorithm overview below.<br />
</p><br />
</p><br />
<br />
<h3>Algorithm overview</h3><br />
<p><br />
C#Sim modeling consists of two major steps:<br />
<ol><br />
<li>Define gene regulatory network structure as a series of related entities (objects): genes to transcribe and translate, promoters, binding sites and transcription factor binding interactions. Specify necessary parameters, such as production rates, degradation rates and promoter binding sites.</li><br />
<br />
<li>Specify the number of simulation steps. At each step of the simulation:<br />
<ol type="I" style="margin-left:15px;"><br />
<li>(optional) introduce input signals and define their actions (e.g. introduce signal 1 at a specific time of the simulation and make it prevent transcription factor from binding);</li><br />
<li>transcribe and translate genes; degrade (i.e. delete) a parameter-specified percentage of produced mRNA and proteins;</li><br />
<li>bind existent transcription factor proteins to their target binding sites; considering binding site capacity (i.e. the number of binding site repeats), uniformly distribute transcription factors among target binding sites;</li><br />
<br />
<li>execute the next simulation step.</li><br />
</ol><br />
</li><br />
</ol><br />
</p><br />
<br />
<br />
<h3>Transcription factor binding</h3><br />
<p><p><br />
Each binding site is modeled as an individual entity. The algorithm, at each simulation step, uniformly distributes transcription factors among their target binding sites based on the amount of each transcription factor available. A binding site entity, <b>B</b>, is defined by the following parameters:<br />
<ul style="margin-left:30px;"><br />
<li>capacity, <b>C</b> (i.e. the number of binding site repeats);</li><br />
<br />
<li>the amount of bound activator, <b>B<sub>A</sub></b> (integer) – how many activator entities are bound to this binding site;</li><br />
<br />
<li>the amount of bound repressor, <b>B<sub>R</sub></b> (integer) – how many repressor entities are bound to this binding site.</li><br />
</ul></p><br />
<p><br />
The sum of all repressors and activators bound to a binding site is never greater than that binding site capacity:<br />
<p><img src="https://static.igem.org/mediawiki/2012/7/71/Svn12_modeling_csim_babr.png"/></p><br />
</p><br />
<br />
<br />
<p><br />
Let <b>A</b> be the amount of activator entities (objects) available for binding and <b>R</b> the amount of repressor entities available for binding. Activators and repressors that competitively bind to a binding site B distribute among the available binding site repeats (specified by capacity C) uniformly, with equal binding affinity, according to the following equations:<br />
<ul style="margin-left:30px;"><br />
<li>if A+R < C, then:<br />
<br/><br />
B<sub>A</sub> = A<br />
<br/><br />
B<sub>R</sub> = R<br />
</li><br/><br />
<br />
<li><br />
if A+R is greater or equal than C, then:<br />
<p><img src="https://static.igem.org/mediawiki/2012/5/5a/Svn12_modeling_csim_bindingsites.png"/></p><br />
</li><br />
</ul><br />
</p><br />
<br />
<p><br />
All values are non-negative integers, i.e. rounding is used to obtain final B<sub>A</sub> and B<sub>R</sub> values. These equations can easily be generalized for a non-competitive binding scenario, when only activators or only repressors bind – in the first case, R is set to 0 (i.e. only activator entities are available); in the second case, A is set to zero (i.e. only repressor entities are available). <br />
</p><br />
<br />
<p><br />
For example, let 10 TAL-A binding site repeats exist, meaning TAL-A binding site capacity is equal to 10. TAL-A:KRAB and TAL-A:VP16 competitively bind for this binding site. Suppose that at a given time the available amount of each of these proteins is 100 units:<br />
<ul style="margin-left:30px;"><br />
<li>TAL-A:KRAB = 100 units</li><br />
<li>TAL-A:VP16 = 100 units</li><br />
</ul><br />
</p><br />
<br />
<p><br />
The amount of TAL-A:VP16 that will bind to the TAL-A binding site is:<br />
<p><img src="https://static.igem.org/mediawiki/2012/9/9a/Svn12_modeling_csim_babr_example1.png"/></p><br />
meaning 5 binding site repeats will become bound by the TAL-A:VP16. Similarly the other 5 will become bound by the TAL-A:KRAB:</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fe/Svn12_modeling_csim_babr_example2.png"/></p><br />
<br />
<p><br />
Thus a uniform distribution was achieved.<br />
</p><br />
<br />
<p><br />
Bound proteins degrade in the same manner as the unbound proteins. It should be noted that with transcription and translation in the context of this algorithm we also mean mRNA and protein degradation. </p><br />
<br />
<br />
<h3>Transcription</h3><br />
<p>The following algorithm steps constitute transcription. For each gene to transcribe at each simulation step:<br />
<ol><br />
<li><br />
count the total amount of repressor and activator entities that are bound to all gene promoter binding sites:<br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/Svn12_modeling_transcription1.png"/></p><br />
<br />
B<sub>Ri</sub> is the amount of repressor bound at binding site i. B<sub>Ai</sub> is the amount of activator bound at binding site i.<br />
</li><br />
<br/><br />
<li><br />
If the promoter is minimal, calculate the amount of mRNA to produce, N'<sub>mRNA</sub>, in this simulation step:<br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fc/Svn12_modeling_transcription2_minimal.png"/></p><br />
Here:<br />
<ul style="margin-left:30px;"><br />
<li>b is inactive (i.e. leaky) transcription rate, specifying mRNA production rate when no activators and no repressors are bound;</li><br />
<br />
<li>k is active (i.e activated) transcription rate and is typically much higher than b; it specifies mRNA production rate when activators are bound;</li><br />
<br />
<li>n and m are transcription factor exponents, specifying the degree of non-linearity.</li><br />
</ul><br />
<br/><p><br />
The structure of this equation is such because we distinguish between three major minimal promoter states:<br />
<ul style="margin-left:30px;"><br />
<li>free (unbound) state, meaning no repressor and no activator entities are bound to corresponding binding sites; in this case, N'<sub>mRNA</sub> will be equal to b (leaky rate);</li><br />
<br />
<li>repressor-bound state, meaning only repressor is bound to corresponding binding sites; in this case, bound repressor can – because of the second term of the equation – decrease the basal rate and hence N'<sub>mRNA</sub>;</li><br />
<br />
<li>activator-bound state, meaning only activator is bound; in this case, the second term of the equation will be equal to zero and (assuming k is much larger than b) N'<sub>mRNA</sub> will be large, indicating activated transcription.</li><br />
</ul> <br />
</p><p><br />
When both activators and repressors are bound to binding sites, none of the three terms will be equal to zero (provided b and k are non-zero values). Because bound repressor count, R<sub>count</sub>, occurs in two terms, while bound activator count, A<sub>count</sub>, only occurs in one term, the effect of bound repressors on N'<sub>mRNA</sub> will be more pronounced compared to the effect of bound activators. This way, a requirement for repression priority over activation (i.e. a small amount of bound repressor significantly reduces expression levels, despite bound activator) is met.</p><br />
</li><br />
<br />
<br/><br />
<li><br />
If the promoter is constitutive, calculate the amount of mRNA to produce, N'<sub>mRNA</sub>, in this simulation step:<br />
<p><img src="https://static.igem.org/mediawiki/2012/f/f5/Svn12_modeling_transcription2_cmv.png"/></p><br />
<p>With our switches in mind, only repressors can bind to constitutive promoter binding sites, hence the equation only has two terms. Here,<br />
<ul style="margin-left:30px;"><br />
<li>k is the active (non-repressed, constitutive) transcription rate, reached only when no repressor entities are bound;<br />
</li><br />
<li>n is an exponent specifying the degree of non-linearity.</li><br />
</ul><br />
Repressor binding can thus decrease N'<sub>mRNA</sub>.<br />
</p><br />
<br />
</li><br />
<br/><br />
<li><p><br />
Randomly increase or decrease calculated N'<sub>mRNA</sub> – this way, stochasticity of transcription is taken into account (e.g. some bound transcription factor can unbind from the binding site). In this step, N'<sub>mRNA</sub> is altered to a new value:</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/3/3e/Svn12_modeling_transcription3_random1.png"/></p><br />
<p><br />
where r is a random integer (uniform distribution is used) from the interval <p><img src="https://static.igem.org/mediawiki/2012/a/a3/Svn12_modeling_transcription3_random2.png" /></p>and t is a real-value parameter between 0 and 1 and specifies the intensity of transcription stochasticity. For our simulations, t was always equal to 0.45.<br />
</p><br />
</li><br />
<br />
<br/><br />
<li><br />
If N<sub>mRNA</sub> was set to 0, make N<sub>mRNA</sub> equal to leaky transcription rate parameter, b, with probability P<sub>L</sub> (probability of leaky expression – equal to 90 % in our simulations). This probability is used to allow more control over the dynamics of leaky expression.<br />
</li><br />
<br />
<br/><br />
<li><br />
Transcribe (generate) N<sub>mRNA</sub> mRNA entities (objects).<br />
</li><br />
<br />
<br/><br />
<li><br />
Degrade m % of mRNA available in the system.<br />
</li><br />
<br />
</ol><br />
</p><br />
<br />
<br />
<h3>Translation</h3><br />
<p><br />
The following algorithm steps constitute translation. For each existing mRNA entity, at each simulation step: <br />
<ol><br />
<li>with a probability of P<sub>T</sub> (we refer to this value as translation probability, or translation effectiveness), generate T<sub>r</sub> protein entities, where T<sub>r</sub> is the translation rate (equal to 1 in all our simulations); mRNA is only translated if the transcription-translation delay has elapsed;</li><br />
<br/><br />
<li>degrade p % of proteins in the system.</li><br />
</ol><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h2><a name="source">Source code</a></h2><br />
<p><br />
Our modeling-related source code can be found <a href="https://2012.igem.org/Team:Slovenia/SourceCode">here</a>.<br />
</p><br />
<br />
<br />
<br />
<br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Andrecut M, Halley JD, Winkler DA, Huang S. (2011) A General Model for Binary Cell Fate Decision Gene Circuits with Degeneracy: Indeterminacy and Switch Behavior in the Absence of Cooperativity. <i>PLoS ONE</i> <b>6</b>, e19358. doi:10.1371/journal.pone.0019358.<br/><br/><br />
<br />
<br />
Kaern M, Blake WJ, Collins JJ. (2003) The engineering of gene regulatory networks. <i>Annual Review of Biomedical Engineering.</i> <b>5</b>, 179-206.<br/><br/><br />
<br />
Kaern M, Elston TC, Blake WJ, Collins JJ. (2005) Stochasticity in gene expression: from theories to phenotypes. <i>Nature.</i> <b>6</b>, 451-464.<br/><br/><br />
<br />
<br />
Ribeiro AS, Lloyd-Price J. (2007) SGN Sim, a Stochastic Genetic Networks Simulator. <i>Bioinformatics.</i> <b>23</b>, 777-779.<br/><br/><br />
<br />
<br />
<br />
Sauro HM. (2012) Enzyme Kinetics for Systems Biology. <i>Future Skill Software.</i><br/><br/><br />
<br />
Siegal-Gaskins D, Mejia-Guerra MK, Smith GD, Grotewold E. (2011) Emergence of Switch-Like Behavior in a Large Family of Simple Biochemical Networks. <i>PLoS Comput Biol</i> <b>7</b>, e1002039. doi:10.1371/journal.pcbi.1002039.<br/><br/><br />
<br />
<br />
Widder S, Macía J, Solé R. (2009) Monomeric Bistability and the Role of Autoloops in Gene Regulation. <i>PLoS ONE</i> <b>4</b>, e5399. doi:10.1371/journal.pone.0005399.<br/><br/><br />
<br />
<br />
</p><br />
<br />
<br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'>Deterministic model of the mutual repressor switch >></a><br />
</b><br />
<br />
<br />
</div><br />
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</body><br />
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<br />
</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/ModelingMethods
Team:Slovenia/ModelingMethods
2013-03-13T21:09:15Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
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<br />
<h1>Modeling methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#intro">Introduction</a><br/></li><br />
<li><a href="#cooperativity">Cooperativity</a></li><br />
<li><a href="#deterministic">Deterministic modeling</a></li><br />
<li><a href="#stochastic">Stochastic modeling</a></li><br />
<li><a href="#quantitative">Quantitative model and stability analysis</a></li><br />
<li><a href="#csim">C#Sim - algorithmic modeling</a></li><br />
<li><a href="#source">Source code</a></li><br />
</ul><br />
</p><br />
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<br />
<br />
<br />
<h2><a name="intro">Introduction</a></h2><br />
<p><br />
In order to examine (i.e. simulate) the proposed genetic switches <i>in silico</i>, different modeling approaches were used. First, a deterministic model based on the probabilistic interpretation of gene regulation was constructed for each type of a genetic switch. Next, a stochastic simulation was performed to take inherent stochastic dynamics of gene expression into account. To further verify the results obtained using these methods, we also developed a quantitative model that builds upon experimental data. Moreover, we developed a modeling algorithm to more explicitly simulate transcription factor binding, considering number of available binding sites and competitive binding. Each modeling approach is discussed in the following sections. We also discuss the notion of cooperativity in the context of bistability.<br />
</p><br />
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<h2><a name="cooperativity">Cooperativity</a></h2><br />
<p><br />
<p><br />
It is often assumed that functional cooperativity (e.g. multimeric regulation) is required for bistability. However, it has been shown theoretically that bistability can emerge in systems without multimeric regulation, provided that at least one regulatory autoloop is present. (Widder et al., 2006). Furthermore, <i>in silico</i> analysis has shown the existence of bistable architectures without the transcription factor cooperativity typically associated with switch-like properties (Siegal-Gaskins et al., 2011). An essential feature of these proposed architectures was the competitive binding of two transcription factors to the promoter.<br />
</p><br />
<p><br />
In terms of modeling, sigmoidal functions (characterizing the rate of change dP/dt) – arising from (Hill) exponents greater than one - are often equated with molecular cooperativity (the way the transcription factor binds to a promoter). However, as non-linearity and multi-stability can arise without assuming molecular cooperativity, it has been suggested that this is not an accurate proposition and that mathematical or functional cooperativity – referring to a sigmoidal function arising from system equations - should not automatically be interpreted as molecular cooperativity (Andrecut et al., 2011). One reason for this is that model equations represent a significant simplification of actual biological dynamics of gene expression, which include a large number of reactions not explicitly considered in modeling, such as reactions describing chromosome opening and transcription initiation.<br />
</p><br />
<br />
<p><br />
For this reasons, we believe that sigmoidal behavior alone – arising in some of our models for transcription factors’ exponent values (non-linearity) greater than 1 - should not by default be interpreted as molecular cooperativity. Thus, in the context of modeling, with the term cooperativity we mean functional cooperativity greater than 1. Functional cooperativity equal to 1 is referred to as no cooperativity.<br />
</p><br />
<br />
<p><br />
Indeed, in case of our positive feedback loop switch – which contains both competitive binding and regulatory autoloops - even deterministic models predict experimentally-verified bistability at low (i.e. close to 1; deterministic fractional occupancy model) or no (quantitative model) functional cooperativity. Our stochastic model of the positive feedback loop switch also predicts bistability is possible without cooperativity.<br />
</p><br />
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</p><br />
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<h2><a name="deterministic">Deterministic modeling</a></h2><br />
<p><br />
<p><br />
Our deterministic models are based on fractional occupancy, a quantity which expresses the degree of saturation at the transcription factor binding site (Sauro, 2012). Fractional occupancy can be expressed as a ratio of active binding site states – i.e. states leading to gene expression – to all possible binding site states:<p><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Svn12_modeling_eqn0.png"/></p><br />
<br/><br />
As such, it can be interpreted as a probability of a promoter being active, or a probability that transcription and/or translation will occur. Gene expression in our model, in turn, is proportional to this probability.<br />
</p><br />
<p><br />
Two main possibilities are distinguished in our models depending on the type of the promoter. In case of a minimal promoter, binding of a transcriptional activator (leading to an active state) is required for transcription initiation. Other states – binding site being free or bound by a repressor - are considered inactive. In case of a constitutive promoter, binding of a transcriptional repressor leads to an inactive state, while unbound (or activator-bound) states are active, hence leading to gene expression.<br />
</p><br />
<p><br />
We assumed large species concentrations compared to the number of available binding sites. We also assumed fast transitions between promoter states, i.e. transcription factor binding and unbinding occurs much faster than transcription/translation, which in this type of deterministic models are represented as a single step of protein production. Another simplification made was a representation of multiple repeats of a binding site as a single binding site (e.g. a specific TAL-A:KRAB binding site may in reality have 10 repeats, while in our models it is represented as a single site). As the purpose of our deterministic models is to provide an approximate, basic characterization of the proposed logic, we consider these simplifications acceptable. Other models, such as C#Sim models (described later), try to formalize some of these aspects in more detail.<br />
</p><br />
<br />
<p><br />
The mathematical framework which describes protein production and into which we incorporate fractional occupancy is represented as a set of ordinary differential equations (ODEs) of the form:<br />
<p><img src="https://static.igem.org/mediawiki/2012/1/16/Svn12_modeling_eqn1.png"/></p><br />
and is roughly based on a framework proposed in (Kaern et al., 2005). Here:<br />
<ul style="margin-left:15px;"><br />
<li>[Protein] is protein concentration at a given time;</li><br />
<li>k is a constant specifying protein production rate in case of an active promoter.</li><br />
<li>kb is a constant specifying the amount of leaky gene expression, meaning protein production that takes place even in the case of inactive (i.e. repressed constitutive or non-activated minimal) promoter.</li><br />
<li>dg is a constant specifying protein degradation rate.</li><br />
<li>P(active) is the probability of a promoter being in an active state – this probability is equal to fractional occupancy f.</li><br />
<li>P(inactive) is the probability of a promoter being inactive and is equal to (1-f).</li><br />
</ul><br />
</p><br />
<br />
<p><br />
No specific quantitative data describing e.g. transcription, translation or degradation rates of TAL effectors was available to us. For this reason, the parameter values (specifying e.g production or degradation rates) in our models were assumed based on commonly accepted propositions, such as that production (i.e. transcription and translation) rates of proteins are (usually) higher than their degradation rates. Parameter values that were used in our simulations should hence only be considered in relative terms (e.g. as protein production to degradation ratios). We also assumed parameter symmetry of different TAL effectors used (TAL-A and TAL-B), because we did not expect that one would have e.g. a production rate significantly higher than the other.<br />
</p><br />
<br />
<p>All deterministic models were implemented in MATLAB.</p><br />
</p><br />
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<h2><a name="stochastic">Stochastic modeling</a></h2><br />
<p><br />
<p><br />
Gene expression is an inherently stochastic process and deterministic simulation is often not satisfactory. The required conditions for the two approaches to be similar are large system size (high mRNA and protein numbers) and fast promoter kinetics (Kaern et al., 2005), but these conditions are not always met. Furthermore, stochastic models can capture the individuality of single cells, as opposed to deterministic models that can only predict the average behavior of a cell population (Kaern et al., 2003). For this reason we performed stochastic simulations of the proposed genetic switches. Our results were obtained using SGN Sim, a stochastic genetic networks simulator (Ribeiro et al., 2007) based on stochastic simulation algorithm (SSA).<br />
</p><br />
<br />
<p><br />
Competitive binding between an activator and a repressor exists in our positive feedback loop switch. This can lead to three major minimal promoter states: the promoter being free (i. e. unbound - neither an activator nor a repressor is bound, zero activation is present), bound by a repressor or bound by an activator. When minimal promoter is unbound, only leaky expression occurs. If bound by an activator, this causes high activated expression levels. Bound repressor causes the expression levels to drop even below the ones achieved for the unbound (non-activated) state.<br />
</p><br />
<br />
<p><br />
Our deterministic models made no clear distinction between free (unbound) minimal promoter state and a repressor-bound state – in both cases inactive promoter state was assumed, leading to only leaky expression. In our stochastic models we improved this by introducing different expression levels for all three states, achieving a more realistic formalization.<br />
</p><br />
<br />
</p><br />
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<br />
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<br />
<h2><a name="quantitative">Quantitative model and stability analysis</a></h2><br />
<p><br />
Based on experimental data we constructed what we refer to as a quantitative model. Please see <a href="https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel">Quantitative and stability model</a> for details. To get a better idea of the functioning of the model, an <a href="https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations">interactive web application</a> was developed.</p><br />
</p><br />
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<br />
<br />
<br />
<h2><a name="csim">C#Sim - algorithmic modeling</a></h2><br />
<p><br />
<p>We developed a new modeling algorithm called C#Sim. The main purpose of this was:<br />
<ul style="margin-left:30px;"><br />
<li>to explicitly model a <b>limited number of binding site repeats;</b></li><br />
<li>to explicitly model <b>competitive binding</b> of activators and repressors;</li><br />
<li>to construct a <b>new modeling approach</b> that incorporates stochasticity of gene expression into an otherwise deterministic approach.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
The assumptions made here were similar to that of deterministic modeling. First, we assumed that high concentrations of mRNA and proteins existed compared to the number of available binding sites that transcription factors could bind to. Second, we assumed that binding of transcription factors to binding sites was much faster than transcription and translation.<br />
</p><br />
<br />
<br />
<p><br />
A feature of C#Sim is that it operates with <b>discrete numbers</b> – at each step of the simulation, mRNA and protein amounts are non-negative integers. Each mRNA and protein molecule is represented as an <b>independent entity</b> – or object - defined by its parameters and interactions. An <b>object-oriented programming approach</b> was used to achieve this. At the moment, C#Sim supports the following entity types, implemented as classes:<br />
<br />
<!-- table --><br />
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<th>mRNA</th><br />
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<td class="normal"><br />
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Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>birth time (integer) – tells at what simulation step this mRNA entity was created (transcribed); used for modeling transcription-translation delay;</li><br />
<li><br />
alive (true/false) – tells if this mRNA is alive and subject to translation (true) or not.</li><br />
</ul><br />
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<th>Protein</th><br />
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Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>birth time (integer) – tells at what simulation step this protein entity was created (translated);</li><br />
<li>alive (true/false) – tells if this protein is alive (true), e.g. it can function as a transcription factor.</li><br />
</ul><br />
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<th>Binding site</th><br />
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<td class="normal"><br />
<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>capacity (integer) – the number of binding site repeats; each repeat can be bound by one protein entity;</li><br />
<li>amount of bound activator (integer) – the number of activator entities bound to this binding site;</li><br />
<li>amount of bound repressor (integer) – the number of repressor entities bound to this binding site.</li><br />
</ul><br />
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</td><br />
</tr> <br />
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<!-- /table --><br />
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<th>Promoter</th><br />
</tr><br />
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<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>promoter type – either minimal or constitutive;</li><br />
<li>list of promoter binding sites;</li><br />
<li>active mRNA transcription rate, which determines the number of mRNA entities to produce at each simulation step for activated promoter (in case of a minimal promoter) or non-repressed promoter (in case of a constitutive promoter);</li><br />
<li>inactive (leaky) mRNA transcription rate, which determines the number of mRNA entities that can be produced even when the promoter is not activated (for minimal promoter), or when the promoter is repressed (for constitutive promoter);</li><br />
<li>the amount of all repressors and activators bound to all promoter binding sites.</li><br />
</ul><br />
<br />
</td><br />
</tr> <br />
</tbody><br />
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<th>Gene</th><br />
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<td class="normal"><br />
<br />
Parameters:<br />
<ul style="margin-left:15px;"><br />
<li>gene's promoter;</li><br />
<li><br />
translation rate, describing the number of proteins produced per each transcribed alive mRNA entity at each simulation step;</li><br />
<li>mRNA and protein degradation rate (i.e. the percentage of mRNA and protein degraded at each simulation step).</li><br />
</ul><br />
<br />
</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<!-- /table --><br />
</p><br />
<br />
<p><br />
Note that the majority of these parameters are computed automatically, not specified by the user (programmer). The parameters specified by the user (when defining the network structure) are: binding sites' capacities, promoter types, list of promoter binding sites, active and inactive transcription rates, a promoter for each gene, translation rates and degradation rates. A delay between transcription and translation can also be specified.<br />
</p><br />
<br />
<p><br />
Using these entities, a hierarchy of gene regulatory network can quickly be constructed. Furthermore, the approach is highly modular and extendible with new dynamics. <!--For examples, please take a look at the <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a>, which contains both our switches implemented in C# language.--><br />
</p><br />
<br />
<p><br />
Other parameters also define the behavior of the mentioned entity types – they are listed and explained in more detail in the algorithm overview below.<br />
</p><br />
</p><br />
<br />
<h3>Algorithm overview</h3><br />
<p><br />
C#Sim modeling consists of two major steps:<br />
<ol><br />
<li>Define gene regulatory network structure as a series of related entities (objects): genes to transcribe and translate, promoters, binding sites and transcription factor binding interactions. Specify necessary parameters, such as production rates, degradation rates and promoter binding sites.</li><br />
<br />
<li>Specify the number of simulation steps. At each step of the simulation:<br />
<ol type="I" style="margin-left:15px;"><br />
<li>(optional) introduce input signals and define their actions (e.g. introduce signal 1 at a specific time of the simulation and make it prevent transcription factor from binding);</li><br />
<li>transcribe and translate genes; degrade (i.e. delete) a parameter-specified percentage of produced mRNA and proteins;</li><br />
<li>bind existent transcription factor proteins to their target binding sites; considering binding site capacity (i.e. the number of binding site repeats), uniformly distribute transcription factors among target binding sites;</li><br />
<br />
<li>execute the next simulation step.</li><br />
</ol><br />
</li><br />
</ol><br />
</p><br />
<br />
<br />
<h3>Transcription factor binding</h3><br />
<p><p><br />
Each binding site is modeled as an individual entity. The algorithm, at each simulation step, uniformly distributes transcription factors among their target binding sites based on the amount of each transcription factor available. A binding site entity, <b>B</b>, is defined by the following parameters:<br />
<ul style="margin-left:30px;"><br />
<li>capacity, <b>C</b> (i.e. the number of binding site repeats);</li><br />
<br />
<li>the amount of bound activator, <b>B<sub>A</sub></b> (integer) – how many activator entities are bound to this binding site;</li><br />
<br />
<li>the amount of bound repressor, <b>B<sub>R</sub></b> (integer) – how many repressor entities are bound to this binding site.</li><br />
</ul></p><br />
<p><br />
The sum of all repressors and activators bound to a binding site is never greater than that binding site capacity:<br />
<p><img src="https://static.igem.org/mediawiki/2012/7/71/Svn12_modeling_csim_babr.png"/></p><br />
</p><br />
<br />
<br />
<p><br />
Let <b>A</b> be the amount of activator entities (objects) available for binding and <b>R</b> the amount of repressor entities available for binding. Activators and repressors that competitively bind to a binding site B distribute among the available binding site repeats (specified by capacity C) uniformly, with equal binding affinity, according to the following equations:<br />
<ul style="margin-left:30px;"><br />
<li>if A+R < C, then:<br />
<br/><br />
B<sub>A</sub> = A<br />
<br/><br />
B<sub>R</sub> = R<br />
</li><br/><br />
<br />
<li><br />
if A+R is greater or equal than C, then:<br />
<p><img src="https://static.igem.org/mediawiki/2012/5/5a/Svn12_modeling_csim_bindingsites.png"/></p><br />
</li><br />
</ul><br />
</p><br />
<br />
<p><br />
All values are non-negative integers, i.e. rounding is used to obtain final B<sub>A</sub> and B<sub>R</sub> values. These equations can easily be generalized for a non-competitive binding scenario, when only activators or only repressors bind – in the first case, R is set to 0 (i.e. only activator entities are available); in the second case, A is set to zero (i.e. only repressor entities are available). <br />
</p><br />
<br />
<p><br />
For example, let 10 TAL-A binding site repeats exist, meaning TAL-A binding site capacity is equal to 10. TAL-A:KRAB and TAL-A:VP16 competitively bind for this binding site. Suppose that at a given time the available amount of each of these proteins is 100 units:<br />
<ul style="margin-left:30px;"><br />
<li>TAL-A:KRAB = 100 units</li><br />
<li>TAL-A:VP16 = 100 units</li><br />
</ul><br />
</p><br />
<br />
<p><br />
The amount of TAL-A:VP16 that will bind to the TAL-A binding site is:<br />
<p><img src="https://static.igem.org/mediawiki/2012/9/9a/Svn12_modeling_csim_babr_example1.png"/></p><br />
meaning 5 binding site repeats will become bound by the TAL-A:VP16. Similarly the other 5 will become bound by the TAL-A:KRAB:</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fe/Svn12_modeling_csim_babr_example2.png"/></p><br />
<br />
<p><br />
Thus a uniform distribution was achieved.<br />
</p><br />
<br />
<p><br />
Bound proteins degrade in the same manner as the unbound proteins. It should be noted that with transcription and translation in the context of this algorithm we also mean mRNA and protein degradation. </p><br />
<br />
<br />
<h3>Transcription</h3><br />
<p>The following algorithm steps constitute transcription. For each gene to transcribe at each simulation step:<br />
<ol><br />
<li><br />
count the total amount of repressor and activator entities that are bound to all gene promoter binding sites:<br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/Svn12_modeling_transcription1.png"/></p><br />
<br />
B<sub>Ri</sub> is the amount of repressor bound at binding site i. B<sub>Ai</sub> is the amount of activator bound at binding site i.<br />
</li><br />
<br/><br />
<li><br />
If the promoter is minimal, calculate the amount of mRNA to produce, N'<sub>mRNA</sub>, in this simulation step:<br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fc/Svn12_modeling_transcription2_minimal.png"/></p><br />
Here:<br />
<ul style="margin-left:30px;"><br />
<li>b is inactive (i.e. leaky) transcription rate, specifying mRNA production rate when no activators and no repressors are bound;</li><br />
<br />
<li>k is active (i.e activated) transcription rate and is typically much higher than b; it specifies mRNA production rate when activators are bound;</li><br />
<br />
<li>n and m are transcription factor exponents, specifying the degree of non-linearity.</li><br />
</ul><br />
<br/><p><br />
The structure of this equation is such because we distinguish between three major minimal promoter states:<br />
<ul style="margin-left:30px;"><br />
<li>free (unbound) state, meaning no repressor and no activator entities are bound to corresponding binding sites; in this case, N'<sub>mRNA</sub> will be equal to b (leaky rate);</li><br />
<br />
<li>repressor-bound state, meaning only repressor is bound to corresponding binding sites; in this case, bound repressor can – because of the second term of the equation – decrease the basal rate and hence N'<sub>mRNA</sub>;</li><br />
<br />
<li>activator-bound state, meaning only activator is bound; in this case, the second term of the equation will be equal to zero and (assuming k is much larger than b) N'<sub>mRNA</sub> will be large, indicating activated transcription.</li><br />
</ul> <br />
</p><p><br />
When both activators and repressors are bound to binding sites, none of the three terms will be equal to zero (provided b and k are non-zero values). Because bound repressor count, R<sub>count</sub>, occurs in two terms, while bound activator count, A<sub>count</sub>, only occurs in one term, the effect of bound repressors on N'<sub>mRNA</sub> will be more pronounced compared to the effect of bound activators. This way, a requirement for repression priority over activation (i.e. a small amount of bound repressor significantly reduces expression levels, despite bound activator) is met.</p><br />
</li><br />
<br />
<br/><br />
<li><br />
If the promoter is constitutive, calculate the amount of mRNA to produce, N'<sub>mRNA</sub>, in this simulation step:<br />
<p><img src="https://static.igem.org/mediawiki/2012/f/f5/Svn12_modeling_transcription2_cmv.png"/></p><br />
<p>With our switches in mind, only repressors can bind to constitutive promoter binding sites, hence the equation only has two terms. Here,<br />
<ul style="margin-left:30px;"><br />
<li>k is the active (non-repressed, constitutive) transcription rate, reached only when no repressor entities are bound;<br />
</li><br />
<li>n is an exponent specifying the degree of non-linearity.</li><br />
</ul><br />
Repressor binding can thus decrease N'<sub>mRNA</sub>.<br />
</p><br />
<br />
</li><br />
<br/><br />
<li><p><br />
Randomly increase or decrease calculated N'<sub>mRNA</sub> – this way, stochasticity of transcription is taken into account (e.g. some bound transcription factor can unbind from the binding site). In this step, N'<sub>mRNA</sub> is altered to a new value:</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/3/3e/Svn12_modeling_transcription3_random1.png"/></p><br />
<p><br />
where r is a random integer (uniform distribution is used) from the interval <p><img src="https://static.igem.org/mediawiki/2012/a/a3/Svn12_modeling_transcription3_random2.png" /></p>and t is a real-value parameter between 0 and 1 and specifies the intensity of transcription stochasticity. For our simulations, t was always equal to 0.45.<br />
</p><br />
</li><br />
<br />
<br/><br />
<li><br />
If N<sub>mRNA</sub> was set to 0, make N<sub>mRNA</sub> equal to leaky transcription rate parameter, b, with probability P<sub>L</sub> (probability of leaky expression – equal to 90 % in our simulations). This probability is used to allow more control over the dynamics of leaky expression.<br />
</li><br />
<br />
<br/><br />
<li><br />
Transcribe (generate) N<sub>mRNA</sub> mRNA entities (objects).<br />
</li><br />
<br />
<br/><br />
<li><br />
Degrade m % of mRNA available in the system.<br />
</li><br />
<br />
</ol><br />
</p><br />
<br />
<br />
<h3>Translation</h3><br />
<p><br />
The following algorithm steps constitute translation. For each existing mRNA entity, at each simulation step: <br />
<ol><br />
<li>with a probability of P<sub>T</sub> (we refer to this value as translation probability, or translation effectiveness), generate T<sub>r</sub> protein entities, where T<sub>r</sub> is the translation rate (equal to 1 in all our simulations); mRNA is only translated if the transcription-translation delay has elapsed;</li><br />
<br/><br />
<li>degrade p % of proteins in the system.</li><br />
</ol><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h2><a name="source">Source code</a></h2><br />
<p><br />
Our modeling-related source code can be found <a href="https://2012.igem.org/Team:Slovenia/SourceCode">here</a>.<br />
</p><br />
<br />
<br />
<br />
<br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Andrecut M, Halley JD, Winkler DA, Huang S. (2011) A General Model for Binary Cell Fate Decision Gene Circuits with Degeneracy: Indeterminacy and Switch Behavior in the Absence of Cooperativity. <i>PLoS ONE</i> <b>6</b>, e19358. doi:10.1371/journal.pone.0019358.<br/><br/><br />
<br />
<br />
Kaern M, Blake WJ, Collins JJ. (2003) The engineering of gene regulatory networks. <i>Annual Review of Biomedical Engineering.</i> <b>5</b>, 179-206.<br/><br/><br />
<br />
Kaern M, Elston TC, Blake WJ, Collins JJ. (2005) Stochasticity in gene expression: from theories to phenotypes. <i>Nature.</i> <b>6</b>, 451-464.<br/><br/><br />
<br />
<br />
Ribeiro AS, Lloyd-Price J. (2007) SGN Sim, a Stochastic Genetic Networks Simulator. <i>Bioinformatics.</i> <b>23</b>, 777-779.<br/><br/><br />
<br />
<br />
<br />
Sauro HM. (2012) Enzyme Kinetics for Systems Biology. <i>Future Skill Software.</i><br/><br/><br />
<br />
Siegal-Gaskins D, Mejia-Guerra MK, Smith GD, Grotewold E. (2011) Emergence of Switch-Like Behavior in a Large Family of Simple Biochemical Networks. <i>PLoS Comput Biol</i> <b>7</b>, e1002039. doi:10.1371/journal.pcbi.1002039.<br/><br/><br />
<br />
<br />
Widder S, Macía J, Solé R. (2009) Monomeric Bistability and the Role of Autoloops in Gene Regulation. <i>PLoS ONE</i> <b>4</b>, e5399. doi:10.1371/journal.pone.0005399.<br/><br/><br />
<br />
<br />
</p><br />
<br />
<br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'>Deterministic model of the mutual repressor switch >></a><br />
</b><br />
<br />
<br />
</div><br />
<!--</div>--><br />
</body><br />
<br />
<br />
</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchCSim
Team:Slovenia/ModelingMutualRepressorSwitchCSim
2013-03-05T13:46:36Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br />
<h1>Modeling - mutual repressor switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch"><b>Deterministic model</b></a></li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchStochastic"><b>Stochastic model</b></a></li><br />
<li><b>C#Sim model</b></li><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
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<h2><a name="summary">C#Sim model of the mutual repressor switch</a></h2><br />
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<p><br />
C#Sim model of the mutual repressor switch suggested, as other models did, that the switch would quickly stop exhibiting bistable behavior with leaky transcription rate increasing past a certain (relatively low) threshold and that only idealized conditions would result in bistability.<br />
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<h2><a name="model">The model</a></h2><br />
<p><br />
The model was constructed in C# programming language by defining objects that represented the switch. <!--See <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a> for complete implementation details.-->See <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">modeling methods</a> for algorithm description.<br />
</p><br />
<br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
State-switching was achieved by introducing state-inducing signals for a certain duration of time. Each signal was modeled as a step function. Each binding site had a capacity equal to 10, to represent 10 binding site repeats. Active transcription rates (k) of all promoters were equal to 200 units. mRNA degradation percentage per simulation step was 0.25 and protein degradation percentage was 0.1.<br />
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Simulation results show reached protein levels (i.e. the amount of protein entities in the system) as a function of time.<br />
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<p><br />
With bistability we mean that even after the removal of inducer signals, the switch remained in the state it had achieved.<br />
</p><br />
<br />
<p><br />
The following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 (with time here we mean simulation step number) to induce stable state 2 (high mCitrine) and removed at <br/>time = 100;</li><br />
<li>signal 1 was introduced at time = 200 to induce stable state 1 (high BFP) and removed at time = 300;</li><br />
<li>signal 2 was again introduced at time = 400 and removed at time = 500;</li><br />
<li>signal 1 was again introduced at time = 600 and removed at time = 700.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
For our first test, leaky transcription of each gene was equal to 15 units (compared to active transcription rate of 200, that means leaking of 7,5%). Translation effectiveness, P<sub>T</sub>, was 25%. All exponents were equal to 1.3. No bistability was exhibited for this scenario, as demonstrated in Figure 1. Increasing transcription factor exponents (n and m) to 3 still produced no bistability. Increasing translation effectiveness (to e.g. 85%) still resulted in no bistable behavior, nor did higher PIP:KRAB or E:KRAB production rate. Bistability was only exhibited for little or no leaky transcription, and even then only for relatively high non-linearity (exponent values of 2 or above). This suggested (as other models did) that leaky expression is highly problematic with the mutual repressor switch and causes it to lose bistable behavior past a certain threshold.<br />
</p><br />
<br />
<p><br />
Figure 2 shows the mutual repressor switch exhibiting bistability when zero leaky expression was present. Transcription factor exponents in this case were equal to 4. Translational effectiveness was 80%. While bistability was exhibited in this case, the reached expression levels could differ significantly between different stable states. Decreasing non-linearity to e.g. 2 lead to loss of bistable behavior.<br />
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<b>Figure 1.</b> The mutual repressor switch exhibited no consistent bistability when leaky transcription was 15 units compared to active transcription rate of 200 units, regardless of non-linearity.<br />
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<b>Figure 2.</b> The mutual repressor switch exhibited bistability in idealized scenario with zero leaky transcription of transcription factors.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'>Deterministic model of the positive feedback loop switch >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim
Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim
2013-03-05T13:34:09Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<br />
<br />
<h1>Modeling - positive feedback loop switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch"><b>Deterministic model</b></a></li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchStochastic"><b>Stochastic model</b></a></li><br />
<li><b>C#Sim model</b></li><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
</ul><br />
</ol><br />
</p><br />
<br />
<br />
<br />
<br />
<h2><a name="summary">C#Sim model of the positive feedback loop switch</a></h2><br />
<!-- summary table --><br />
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<td class="summary" style="font-size:110%;"><br />
<p><br />
C#Sim model of the positive feedback loop switch, like other modeling approaches, showed that this switch was much more robust than the mutual repressor switch. The positive feedback loop switch would exhibit bistability even for low transcription factor exponent values, such as 1.1 - much lower than required for bistability of the mutual repressor switch. It again proved tolerant to leaky production of transcription factors and exhibited bistability even for low translation effectiveness (e.g. 25%).<br />
</p><p><br />
Decreasing translation effectiveness required a relatively slight increase in transcription factor exponent values for bistability to occur.<br />
</p><br />
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<br />
<br />
<h2><a name="model">The model</a></h2><br />
<p><br />
The model was constructed in C# programming language by defining objects that represented the switch. <!--See <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a> for complete implementation details.-->See <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">modeling methods</a> for algorithm description.<br />
</p><br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
State-switching was achieved by introducing state-inducing signals for a certain duration of time. Each signal was modeled as a step function. Each binding site had a capacity equal to 10, to represent 10 binding site repeats. Active transcription rates (k) of all promoters were equal to 200 units. mRNA degradation percentage per simulation step was 0.25 and protein degradation percentage was 0.1.<br />
</p><br />
<br />
<p><br />
Simulation results show reached protein levels (i.e. the amount of protein entities in the system) as a function of time.<br />
</p><br />
<br />
<p><br />
In our first test, the following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 to induce stable state 2 (high mCitrine) and removed at time = 100;</li><br />
<li>signal 1 was introduced at time = 200 to induce stable state 1 (high BFP) and removed at time = 300;</li><br />
<li>signal 2 was again introduced at time = 400 and removed at time = 500;</li><br />
<li>signal 1 was again introduced at time = 600 and removed at time = 700.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Leaky expression (b) of each gene was equal to 15 units (compared to active transcription rate of 200, that means leaking of 7,5%). Exponent values (m and n - see <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods">Modeling methods</a> for description) were equal to 1.3. Translation effectiveness was 25%. While the mutual repressor switch didn't exhibit bistability for this parameter values, the positive feedback loop switch did, as shown in figure 1.<br />
</p><br />
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<b>Figure 1. </b>The positive feedback loop switch transitioning between stable states for parameter values that did not result in bistability of the mutual repressor switch (here, transcription factor exponents were equal to 1.3 and leaky transcription rates were 15 units).<br />
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<p><br />
For our next tests, the following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 (with time here we mean simulation step number) to induce stable state 2 (high mCitrine) and removed at <br/>time = 100;</li><br />
<li>signal 1 was introduced at time = 400 to induce stable state 1 (high BFP) and removed at time = 500;</li><br />
<li>signal 2 was again introduced at time = 800 and removed at time = 900;</li><br />
<li>signal 1 was again introduced at time = 1200 and removed at time = 1300.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Exponents were equal to 1.1. Leaky production rate of all proteins was again 15 units. Translation effectiveness was 90%. Bistability was exhibited, as shown in figure 2. Decreasing translation effectiveness required a slight increase in exponent values for bistability to occur. Figure 3 shows that bistability was exhibited fo exponent values of 1.3 when translation effectiveness was reduced to 40%. Figure 4 shows the switch exhibiting bistability for exponent values equal to 1.3 with leaky transcription rate equal to 15 units and translation effectiveness equal to 100%.<br />
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<b>Figure 2.</b> The positive feedback loop switch exhibited bistability for exponent values of 1.1 when translation effectiveness was 90%, despite leaky expression of 15 units for all genes.<br />
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<b>Figure 3.</b> The positive feedback loop switch exhibited bistability for exponent values of 1.3 when translation effectiveness was 40%.<br />
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<b>Figure 4.</b> The positive feedback loop switch exhibiting bistability for translation effectiveness of 100%.<br />
</td></tr><br />
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<br />
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<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'>Experimental model >></a><br />
</b><br />
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</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/SourceCode
Team:Slovenia/SourceCode
2013-03-05T13:17:37Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<!-- end main menu --><br />
<br />
<br />
</div> <!-- end menu --><br />
<br />
<div id="main"><br />
<br/><br />
<br />
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<a href="https://2012.igem.org/Main_Page"><br />
<div id="dummy" style="background-color:transparent; position:absolute; left:870px; top:25px; width:115px; height:80px; z-index:100; opacity:0.0;"><br />
</div><br />
</a><br />
<br />
<br />
<h1>Source code</h1><br />
<br />
<h3>MATLAB - deterministic models</h3><br />
<p><br />
MATLAB source code of our deterministic models. Requires <a href="http://www.mathworks.com/matlabcentral/fileexchange/23629">export_fig</a> to work.<br />
<br/><a href="https://static.igem.org/mediawiki/2012/7/7f/MATLAB_deterministic_models_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>SGN Sim - stochastic models</h3><br />
<p><br />
<a href="http://www.cs.tut.fi/~sanchesr/SGN/SGNSim.html">SGN Sim</a> files with our stochastic model.<br/><br />
<a href="https://static.igem.org/mediawiki/2012/3/30/SGNSIM_stochastic_models_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<br />
<br />
<h3>Experimental model</h3><br />
<p><br />
Python source code for the Exeprimental model.<br/> <br />
Requires: <a href="http://www.python.org/">Python</a> interpreter, <a href="http://numpy.scipy.org">NumPy</a> module, <a href="http://www.scipy.org">SciPy</a> Module.<br/><br />
<a href="https://static.igem.org/mediawiki/2012/e/e4/Svn12_ExperimentalModel.zip">DOWNLOAD</a><br/><br />
<br />
</p><br />
<br />
<h3>Pharmacokinetic real-time 3D simulation</h3><br />
<p><br />
Executables (Windows) and C++ source code for our pharmacokinetic 3D visualuzation. Requires <a href="http://freeglut.sourceforge.net/">freeglut library</a>. Hosted remotely due to large file size.<br/><br />
<a href="http://igem2012.fri.uni-lj.si/PK3D_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<br />
</div><br />
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</body><br />
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</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/Slovenian
Team:Slovenia/Slovenian
2012-10-27T03:48:16Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<strong style="font-size:120%;">Izziv</strong><br/><br />
<br /><br />
<p>Biološka zdravila, kot so hormoni, encimi, citokini ter protitelesa, se vse bolj pogosto uporabljajo za zdravljenje različnih bolezni. Ker poteka zdravljenje z biološkimi zdravili sistemsko (npr. zdravilo se injicira v kri, tako da doseže celotno telo, ne le obolelega tkiva), ima mnogokrat lahko stranske učinke. Poleg tega cena bioloških zdravil predstavlja hudo breme zdravstveni blagajni.</p><br />
<p>Naš cilj je bil razviti varen in cenovno ugoden sistem za produkcijo ter dostavo bioloških zdravil, ki bi izboljšal kakovost življenja mnogim pacientom. Zaradi lokalne aplikacije bi bili neželeni stranski učinki zmanjšani, pacienti pa bi se lažje držali režima terapije. Prav tako bi naš sistem zmanjšal število invazivnih zdravniških posegov, hkrati pa omogočil napredno večstopenjsko zdravljenje.</p><br />
<p>Sistem za produkcijo in dostavo bioloških zdravil smo načrtovali v obliki mikroenkapsuliranih sesalskih celic, ki bi jih lahko kontrolirali ter jih usmerili v produkcijo različnih zdravil. Tak sistem bi bil zanesljiv, cenovno učinkovit in varen.</p><br />
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<a href="https://2012.igem.org/Team:Slovenia/TheSwitch"><img class="pic" src="https://static.igem.org/mediawiki/2012/3/38/Svn12_stikalo.png" width="300" height="300" /></a><br />
<div class="ttip"><br />
<strong>Stikalo: nov tip ortogonalnega preklopnega stikala</strong><br />
<p>S pomočjo DNA-vezavnih proteinov smo pripravili <b>nov tip bistabilnega preklopnega <br />
stikala za sesalske celice</b>, ki omogoča tudi razširitev v več ortogonalnih stikal <br />
in na ta način pripravo kompleksnih logičnih naprav. Ugotovili smo, da je <b>klasična<br />
shema preklopnih stikal neučinkovita</b>, če uporabimo kot represorje efektorje TAL, <br />
saj se ti vežejo na DNA nekooperativno kot monomeri. Ta problem smo rešili tako, da <br />
smo pripravili stikalo, sestavljeno iz para medsebojnih represorjev (TAL:KRAB), <br />
sklopljenega s parom aktivatorjev (TAL:VP16), ki tvorita <b>pozitivno povratno zanko</b>. <br />
Eksperimentalno smo potrdili, da lahko takšen sistem v sesalskih celicah vodi do bistabilnega stanja, <br />
ki ga lahko nadzorujemo s pomočjo nizkomolekularnih induktorjev. <a href="https://2012.igem.org/Team:Slovenia/TheSwitch">Preberi več...</a></p><br />
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<td class="splash"><br />
<a href="https://2012.igem.org/Team:Slovenia/SafetyMechanisms"><img class="pic" src="https://static.igem.org/mediawiki/2012/f/f8/Svn12_varnost.png" width="300" height="300"/></a><br />
<div class="ttip"><br />
<strong>Varnost</strong><br />
<p>Da bi bil naš sistem mikroenkapsuliranih sesalskih celic povsem varen za paciente, smo načrtovali več varnostnih mehanizmov. Tako se bodo alginatne mikrokapsule po koncu terapije lahko varno razgradile, v terapevtskih celicah pa se bo nadzorovano sprožila apoptoza oz. programirana celična smrt. Za vsak slučaj, da ne bi katera od celic nenadzorovano ušla iz mikrokapsule, pa smo celice opremili tudi z oznako, ki omogoča učinkovito odstranitev terapevtskih celic s pomočjo telesu lastnih imunskih celic, t.i. naravnih ubijalk. <a href="https://2012.igem.org/Team:Slovenia/SafetyMechanisms">Preberi več...</a></p><br />
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<td class="splash"><br />
<a href="https://2012.igem.org/Team:Slovenia/Implementation"><img class="pic" src="https://static.igem.org/mediawiki/2012/4/42/Svn12_vgradnja.png" width="300" height="300" /></a><br />
<div class="ttip"><br />
<strong>Uporaba: napredno zdravljenje različnih bolezni</strong><br />
<p>Naš sistem smo oblikovali za zdravljenje hepatitisa C ter srčne ishemije. Uporabili smo pet različnih terapevtskih proteinov, za katere je naš farmakokinetični model pokazal, da bi njihova uporaba lahko zmanjšala neželene stranske učinke in izboljšala učinkovitost terapije. Preklop med produkcijo protivirusnih oz. protivnetnih učinkovin ter produkcijo učinkovin, ki bi pospešile regeneracijo obolelega tkiva, bi od zunaj kontroliral zdravnik s pomočjo nizkomolekularnih induktorjev. <a href="https://2012.igem.org/Team:Slovenia/Implementation">Preberi več...</a></p><br />
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<a href="https://2012.igem.org/Team:Slovenia/Modeling"><img class="pic" src="https://static.igem.org/mediawiki/2012/d/da/Svn12_modeliranje.png" width="300" height="300" /></a><br />
<div class="ttip2"><br />
<strong>Modeliranje: analiza genskega stikala ter farmakokinetike</strong><br />
<p>Obsežno matematično modeliranje je pokazalo, da klasično preklopno stikalo ni stabilno brez kooperativnosti. Hkrati je modeliranje pokazalo, da uporaba modularnih DNA-vezavnih transkripcijskih regulatorjev, ki tvorijo dvojno pozitivno povratno zanko, izboljša robustnost stikala. Takšna topologija ne zahteva kooperativnosti, saj nelinearnost nastane zaradi pozitivne povratne zanke. Farmakokinetični model lokalne dostave terapevtika z mikroenkapsuliranimi celicami predvidi, da bi predlagana terapija lahko zmanjšala neželene stranske učinke. <a href="https://2012.igem.org/Team:Slovenia/Modeling">Preberi več...</a></p><br />
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</td><br />
<br />
<td class="splash"><br />
<a href="https://2012.igem.org/Team:Slovenia/Society"><img class="pic" src="https://static.igem.org/mediawiki/2012/0/09/Svn12_druzba.png" width="300" height="300" /></a><br />
<div class="ttip2"><br />
<strong>Družba: vključenost vseh, na katere lahko sintezna biologija vpliva</strong><br />
<p>Pri pripravi našega projekta smo želeli upoštevati mnenja ljudi iz različnih sfer, tako zdravnikov kot pacientov, pripravljalcev in nadzornikov zakonodaje, splošne javnosti, medijev ter znanstvenikov. Z njimi smo se pogovarjali o različnih vidikih našega projekta ter o medicinskih aplikacijah sintezne biologije, saj bodo njihovi pogledi pomembno vplivali na uspešno uvedbo sintezne biologije v klinično prakso. Sintezno biologijo ter njene medicinske in ostale potenciale smo želeli predstaviti tudi mlajšim generacijam, zato smo vspostavili stike tudi z različnimi srednjimi šolami. <a href="https://2012.igem.org/Team:Slovenia/Society">Preberi več...</a></p><br />
</div><br />
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<br />
<td class="splash"><br />
<a href="https://2012.igem.org/Team:Slovenia/SocietyEthics"><img class="pic" src="https://static.igem.org/mediawiki/2012/4/42/Svn12_obeti.png" width="300" height="300" /></a><br />
<div class="ttip2"><br />
<strong>Obeti: prispevek k drugim aplikacijam ter načrti za prihodnost</strong><br />
<ul style="padding-left:30px;"><br />
<li>Varnostni mehanizmi (oznaka, ki olajša nadzor imunskemu sistemu, nadzorovana razgradnja kapsul ter inducirana smrt terapevtskih celic) omogočajo uporabo tudi za druge terapevtske namene,</li><br />
<li>namen imamo pripraviti stabilne celične linije, ki bi imele vključene vse komponente stikala z različnimi terapevtskimi učinkovinami in varnostnimi mehanizmi,</li><br />
<li>načrtovana ortogonalna logika, ki temelji na transkripcijskih faktorjih povezanih z elementi TAL, bo omogočila pripravo desetine ali stotine različnih stikal in doseganje multistabilnih stanj ter drugih kompleksnih logičnih naprav,</li><br />
<li>pričeti želimo s prvimi <i>in vivo</i> poskusi na področju ishemije in celjenja ran.</li><br />
</ul><br />
<p><a href="https://2012.igem.org/Team:Slovenia/Implementation">Preberi več...</a></p><br />
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<strong style="font-size:120%;">Povzetek za nestrokovnjake</strong><br/><br />
<br /><br />
<p>Biološka zdravila se čedalje pogostje uporabljajo za zdravljenje različnih bolezni, saj imajo zaradi bolj specifične narave njihovega delovanja mnogo prednosti pred običajnimi zdravili. Biološka zdravila se ponavadi dajejo sistemsko, zaradi česar se zdravilo razporedi bolj ali manj po celem telesu, čeprav bi bilo mnogokrat zaželeno, da bi delovalo le na določeno tarčno tkivo oz. organ. Zato ima običajna oblika zdravljenja mnogokrat nevarne stranske učinke, terapija pa zahteva višje doze zdravila, kar poveča tudi ceno zdravljenja. Naša ideja za rešitev tega problema je, da bi direktno v obolelo tkivo vstavili celice, ki bi proizvajale biološko zdravilo. Te terapevtske celice bi bile varno spravljene v zaščitni mikrokapsuli, ki bi preprečila širjenje teh celic po telesu, hkrati pa bi jih zaščitila pred imunskim sistemom bolnika, ki bi sicer napadel in uničil telesu tuje celice. Razvili smo biološko napravo, ki terapevtskim celicam omogoča, da proizvedejo različna zdravila, zdravnik pa bi lahko nadzoroval, katero zdravilo bi se proizvajalo glede na fazo bolezni. Terapevtske celice smo prilagodili z namenom zdravljenja hepatitisa C, tako da lahko proizvajajo biološko zdravilo s protivirusnim učinkom, nato pa še zdravilo, ki pospeši regeneracijo jeter. Z namenom terapije po srčnem infarktu pa smo terapevtske celice prilagodili tako, da njihovo delovanje zmanjša škodljivo vnetje ter pospeši tvorbo novih krvnih žil v okolici prizadetega tkiva. Ob koncu terapije (po potrebi pa tudi vmes) lahko zdravnik preko zunanjega signala sproži samouničenje terapevtskih celic in mikrokapsul. Verjamemo, da je naš sistem varen in učinkovit ter da bi se ga dalo prilagoditi za zdravljenje različnih tipov bolezni.</p><br />
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<strong>Dosežki (bolj podrobno):</strong><br />
<ul style="padding-left: 30px;"><br />
<li>Prvi smo eksperimentalno pokazali delovanje bistabilnega preklopnega stikala v sesalskih celicah, ki temelji na nekooperativnih DNA-vezavnih proteinih, ter bistabilnega stikala, ki temelji na načrtovanih DNA-vezavnih proteinih.</li><br />
<li>V sesalskih celicah smo pripravili in preizkusili bistabilno preklopno stikalo, osnovano na ortogonalnih TAL-represorjih in aktivatorjih.</li><br />
<li>Matematično modeliranje je pokazalo izboljšanje robustnosti stikala, osnovanega na pozitivni povratni zanki, tudi ob upoštevanju puščanja transkripcije.</li><br />
<li>V mikroenkapsulirane sesalske celice smo vgradili tri varnostne mehanizme: <br />
<ul class="circle" style="padding-left: 50px;"><br />
<li>oznako, ki omogoča uničenje iz mikrokapsul pobeglih celic s pomočjo celic naravnih ubijalk;</li><br />
<li>sekretorno alginat liazo, ki razgradi alginatne mikrokapsule;</li><br />
<li>sistem za sprožitev programirane celične smrti terapevtskih celic ob dodatku ganciklovirja.</li><br />
</ul><br />
</li><br />
<li>Predvideli smo kombinacijo dveh bioloških zdravil (interferona alfa ter rastnega faktorja hepatocitov) za zdravljenje hepatitisa C, saj lahko na tak način v prvi fazi zdravljenja inaktiviramo virus, v naslednji fazi pa spodbudimo regeneracijo jeter.</li><br />
<li>Za zdravljenje ishemije smo predvideli kombinacijo večih bioloških zdravil (anakinre, vaskularnega endotelijskega rastnega faktorja, rastnega faktorja iz trombocitov), da bi na tak način najprej zmanjšali neželeno vnetje, nato pa v drugi fazi zdravljenja pospešili nastajanje novih krvnih žil.</li><br />
<li>Pripravili smo 89 novih konstruktov DNA, ki so kompatibilni s sintezno-biološkim standardom »BioBrick« in smo jih uporabili v načrtovanih bioloških napravah.</li><br />
<li>Izboljšali smo enega od že obstoječih standardnih "BioBrick" konstruktov DNA.</li><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim
Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim
2012-10-26T21:50:12Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
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<h1>Modeling - positive feedback loop switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch"><b>Deterministic model</b></a></li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchStochastic"><b>Stochastic model</b></a></li><br />
<li><b>C#Sim model</b></li><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
</ul><br />
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<h2><a name="summary">C#Sim model of the positive feedback loop switch</a></h2><br />
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<p><br />
C#Sim model of the positive feedback loop switch, like other modeling approaches, showed that this switch was much more robust than the mutual repressor switch. The positive feedback loop switch would exhibit bistability even for low transcription factor exponent values, such as 1.1 - much lower than required for bistability of the mutual repressor switch. It again proved tolerant to leaky production of transcription factors and exhibited bistability even for low translation effectiveness (e.g. 25%).<br />
</p><p><br />
Decreasing translation effectiveness required a relatively slight increase in transcription factor exponent values for bistability to occur.<br />
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<h2><a name="model">The model</a></h2><br />
<p><br />
The model was constructed in C# programming language by defining objects that represented the switch. See <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a> for complete implementation details. See <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">modeling methods</a> for algorithm description.<br />
</p><br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
State-switching was achieved by introducing state-inducing signals for a certain duration of time. Each signal was modeled as a step function. Each binding site had a capacity equal to 10, to represent 10 binding site repeats. Active transcription rates (k) of all promoters were equal to 200 units. mRNA degradation percentage per simulation step was 0.25 and protein degradation percentage was 0.1.<br />
</p><br />
<br />
<p><br />
Simulation results show reached protein levels (i.e. the amount of protein entities in the system) as a function of time.<br />
</p><br />
<br />
<p><br />
In our first test, the following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 to induce stable state 2 (high mCitrine) and removed at time = 100;</li><br />
<li>signal 1 was introduced at time = 200 to induce stable state 1 (high BFP) and removed at time = 300;</li><br />
<li>signal 2 was again introduced at time = 400 and removed at time = 500;</li><br />
<li>signal 1 was again introduced at time = 600 and removed at time = 700.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Leaky expression (b) of each gene was equal to 15 units (compared to active transcription rate of 200, that means leaking of 7,5%). Exponent values (m and n - see <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods">Modeling methods</a> for description) were equal to 1.3. Translation effectiveness was 25%. While the mutual repressor switch didn't exhibit bistability for this parameter values, the positive feedback loop switch did, as shown in figure 1.<br />
</p><br />
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<b>Figure 1. </b>The positive feedback loop switch transitioning between stable states for parameter values that did not result in bistability of the mutual repressor switch (here, transcription factor exponents were equal to 1.3 and leaky transcription rates were 15 units).<br />
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<p><br />
For our next tests, the following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 (with time here we mean simulation step number) to induce stable state 2 (high mCitrine) and removed at <br/>time = 100;</li><br />
<li>signal 1 was introduced at time = 400 to induce stable state 1 (high BFP) and removed at time = 500;</li><br />
<li>signal 2 was again introduced at time = 800 and removed at time = 900;</li><br />
<li>signal 1 was again introduced at time = 1200 and removed at time = 1300.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Exponents were equal to 1.1. Leaky production rate of all proteins was again 15 units. Translation effectiveness was 90%. Bistability was exhibited, as shown in figure 2. Decreasing translation effectiveness required a slight increase in exponent values for bistability to occur. Figure 3 shows that bistability was exhibited fo exponent values of 1.3 when translation effectiveness was reduced to 40%. Figure 4 shows the switch exhibiting bistability for exponent values equal to 1.3 with leaky transcription rate equal to 15 units and translation effectiveness equal to 100%.<br />
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<b>Figure 2.</b> The positive feedback loop switch exhibited bistability for exponent values of 1.1 when translation effectiveness was 90%, despite leaky expression of 15 units for all genes.<br />
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<b>Figure 3.</b> The positive feedback loop switch exhibited bistability for exponent values of 1.3 when translation effectiveness was 40%.<br />
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<b>Figure 4.</b> The positive feedback loop switch exhibiting bistability for translation effectiveness of 100%.<br />
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<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'>Experimental model >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchStochastic
Team:Slovenia/ModelingPositiveFeedbackLoopSwitchStochastic
2012-10-26T21:41:56Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
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<br />
<h1>Modeling - positive feedback loop switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch"><b>Deterministic model</b></a><br />
</li><br />
<li><b>Stochastic model</b><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim"><b>C#Sim model</b></a></li><br />
</ol><br />
</p><br />
<br />
<br />
<br />
<h2><a name="summary">Stochastic model of the positive feedback loop switch</a></h2><br />
<!-- summary table --><br />
<table class="summary"><br />
<tr class="summary"><br />
<td class="summary" style="font-size:110%;"><br />
<p><br />
Stochastic simulation, in agreement with the deterministic analysis, proved that the positive feedback loop switch was more robust than the mutual repressor switch. It exhibited bistability without cooperativity (i.e. cooperativity equal to 1) even for leaky expression that caused the mutual repressor switch to lose bistable behavior. Higher cooperativity further increased the robustness and leaky expression tolerance, allowing for higher expression levels to be reached.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<!-- /summary table --><br />
<br />
<br />
<br />
<h2><a name="model">The model</a></h2><br />
<p><br />
The basis for the stochastic simulation of the positive feedback loop switch was the following set of reactions that describe the dynamics of the switch:<br />
</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/0/05/Svn12_m_pfs_stoch1.PNG" /></p><br/><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/93/Svn12_m_pfs_stoch2.PNG" /></p><br/><br />
<p><img src="https://static.igem.org/mediawiki/2012/3/3e/Svn12_m_pfs_stoch3.PNG" /></p><br/><br />
<p><img src="https://static.igem.org/mediawiki/2012/5/54/Svn12_m_pfs_stoch4.PNG" /></p><br/><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/6e/Svn12_m_pfs_stoch5.PNG" /></p><br/><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/82/Svn12_m_pfs_stoch6.PNG" /></p><br/><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ea/Svn12_m_pfs_stoch7.PNG" /></p><br />
<br />
<p><br />
Here:<br />
<ul style="margin-left:30px;"><br />
<li>Pro1 is construct 1 promoter (i.e. promoter 1 - minimal);</li><br />
<li>Pro2 is construct 2 promoter (i.e. promoter 2 - minimal);</li><br />
<li>Pro3 is construct 3 promoter (i.e. promoter 3 - constitutive);</li><br />
<li>Pro4 is construct 4 promoter (i.e. promoter 4 - constitutive);</li><br />
<li>Pro5 is construct 5 promoter (i.e. promoter 5 - constitutive);</li><br />
</ul><br />
</p><br />
<br />
<p>It is implicitly assumed that when both activator (TAL:VP16) and repressor (TAL:KRAB) bind to the promoter, the effect of the repressor will be more significant.</p><br />
<br />
<p><br />
See <a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchStochastic#model">the mutual repressor switch stochastic model</a> for additional description.<br />
</p><br />
<br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
The same assumptions apply as for the mutual repressor switch simulation. Detailed parameter values for each simulation can be found in the corresponding simulation files that can be found <a href="https://2012.igem.org/Team:Slovenia/SourceCode">here</a>.<br />
</p><br />
<br />
<p><br />
Figure 1 shows the result of the first simulation, where zero leaky expression and no cooperativity were assumed. Bistability was exhibited, just like it was in the deterministic simulation. The state-switching scenario used was the same as for the mutual repressor switch:<br />
<ul style="margin-left:30px;"><br />
<li>at time = 500, signal 2 was introduced, inducing stable state 2 (high mCitrine state);</li><br />
<li>at time = 2500, signal 1 was introduced, inducing stable state 1 (high BFP state);</li><br />
<li>at time = 4500, signal 2 was introduced, switching the system to stable state 2;</li><br />
<li>at time = 6500, signal 1 was introduced, switching the system to stable state 1.</li><br />
</ul><br />
</p><br />
<br />
<br />
<p><br />
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<b>Figure 1.</b> Positive feedback loop switch transitioning between stable states. No cooperativity and zero leaky expression were assumed.<br />
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<p><br />
While the introduction of leaky expression of 0.03 for each gene caused the mutual repressor switch to stop exhibiting bistable behavior, this was not the case for the positive feedback loop switch. Here, while the stable-state levels dropped moderately, bistability was observed in such case – shown in Figure 2 - even with no cooperativity (i.e. cooperativity equal to 1). This was in agreement with the deterministic model.<br />
</p><br />
<br />
<p><br />
The simulation showed that the constitutive promoters leakage was more detrimental to bistability than the minimal promoters leakage. The leaky expression tolerance depended on both production and degradation rates (with production to degradation rate ratios too high or too low resulting in no bistable behavior). Figure 3 shows the positive feedback switch exhibiting bistability without cooperativity for minimal promoters leaky transcription of 10% and constitutive promoters leaky transcription of 5%. The mutual repressor switch did not exhibit bistability for these parameters.<br />
</p><br />
<br />
<p><br />
When leaky expression was above a certain threshold (depending on other parameter values), cooperativity was required for bistability. Figure 4 shows a case where leaky expression of 0.08 did not result in bistability loss if cooperativity was equal to 2.<br />
Increasing leaky expression resulted in lower stable-state levels. Higher cooperativity improved this, allowing high (maximal) levels to be reached. Cooperativity being too high, like for the mutual repressor switch, caused the loss of bistability.<br />
</p><br />
<br />
<br />
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<b>Figure 2.</b> Positive feedback loop switch exhibited bistability when leaky expression was equal to 0.03 – the same value that caused the mutual repressor switch to lose bistability - despite no cooperativity.<br />
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<b>Figure 3.</b> Positive feedback loop switch exhibited bistability without cooperativity for minimal promoters leaky transcription equal to 10% and constitutive promoters leaky transcription equal to 5%. Protein production rates (i.e. rates for non-repressed constitutive promoters and activated minimal promoters) were 1.0 and protein degradation rates were 0.15. Increasing protein degradation rates from 0.1 to 0.15 improved the tolerance to leakage.<br />
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<b>Figure 4.</b> Bistability was observed for leaky expression of all genes equal to 0.08 as long as high-enough cooperativity was used. Here, cooperativity was equal to 2, with protein production rates equal to 1.0 and degradation rates equal to 0.1.<br />
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<br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim'>C#Sim model of the positive feedback loop switch >></a><br />
</b><br />
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</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchCSim
Team:Slovenia/ModelingMutualRepressorSwitchCSim
2012-10-26T21:19:17Z
<p>Dusanv: </p>
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</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<br />
<h1>Modeling - mutual repressor switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch"><b>Deterministic model</b></a></li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchStochastic"><b>Stochastic model</b></a></li><br />
<li><b>C#Sim model</b></li><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
</ul><br />
</ol><br />
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<br />
<br />
<h2><a name="summary">C#Sim model of the mutual repressor switch</a></h2><br />
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<p><br />
C#Sim model of the mutual repressor switch suggested, as other models did, that the switch would quickly stop exhibiting bistable behavior with leaky transcription rate increasing past a certain (relatively low) threshold and that only idealized conditions would result in bistability.<br />
</p><br />
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<h2><a name="model">The model</a></h2><br />
<p><br />
The model was constructed in C# programming language by defining objects that represented the switch. See <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a> for complete implementation details. See <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">modeling methods</a> for algorithm description.<br />
</p><br />
<br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
State-switching was achieved by introducing state-inducing signals for a certain duration of time. Each signal was modeled as a step function. Each binding site had a capacity equal to 10, to represent 10 binding site repeats. Active transcription rates (k) of all promoters were equal to 200 units. mRNA degradation percentage per simulation step was 0.25 and protein degradation percentage was 0.1.<br />
</p><br />
<br />
<p><br />
Simulation results show reached protein levels (i.e. the amount of protein entities in the system) as a function of time.<br />
</p><br />
<br />
<p><br />
With bistability we mean that even after the removal of inducer signals, the switch remained in the state it had achieved.<br />
</p><br />
<br />
<p><br />
The following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 (with time here we mean simulation step number) to induce stable state 2 (high mCitrine) and removed at <br/>time = 100;</li><br />
<li>signal 1 was introduced at time = 200 to induce stable state 1 (high BFP) and removed at time = 300;</li><br />
<li>signal 2 was again introduced at time = 400 and removed at time = 500;</li><br />
<li>signal 1 was again introduced at time = 600 and removed at time = 700.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
For our first test, leaky transcription of each gene was equal to 15 units (compared to active transcription rate of 200, that means leaking of 7,5%). Translation effectiveness, P<sub>T</sub>, was 25%. All exponents were equal to 1.3. No bistability was exhibited for this scenario, as demonstrated in Figure 1. Increasing transcription factor exponents (n and m) to 3 still produced no bistability. Increasing translation effectiveness (to e.g. 85%) still resulted in no bistable behavior, nor did higher PIP:KRAB or E:KRAB production rate. Bistability was only exhibited for little or no leaky transcription, and even then only for relatively high non-linearity (exponent values of 2 or above). This suggested (as other models did) that leaky expression is highly problematic with the mutual repressor switch and causes it to lose bistable behavior past a certain threshold.<br />
</p><br />
<br />
<p><br />
Figure 2 shows the mutual repressor switch exhibiting bistability when zero leaky expression was present. Transcription factor exponents in this case were equal to 4. Translational effectiveness was 80%. While bistability was exhibited in this case, the reached expression levels could differ significantly between different stable states. Decreasing non-linearity to e.g. 2 lead to loss of bistable behavior.<br />
</p><br />
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<b>Figure 1.</b> The mutual repressor switch exhibited no consistent bistability when leaky transcription was 15 units compared to active transcription rate of 200 units, regardless of non-linearity.<br />
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<b>Figure 2.</b> The mutual repressor switch exhibited bistability in idealized scenario with zero leaky transcription of transcription factors.<br />
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<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'>Deterministic model of the positive feedback loop switch >></a><br />
</b><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchStochastic
Team:Slovenia/ModelingMutualRepressorSwitchStochastic
2012-10-26T21:16:03Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
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<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
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<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<h1>Modeling - mutual repressor switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch"><b>Deterministic model</b></a><br />
</li><br />
<li><b>Stochastic model</b><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchCSim"><b>C#Sim model</b></a></li><br />
</ol><br />
</p><br />
<br />
<br />
<br />
<h2><a name="summary">Stochastic model of the mutual repressor switch</a></h2><br />
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<p><br />
Stochastic simulation revealed that the mutual repressor switch exhibited bistability only as long as there was no leaky expression present. Otherwise, cooperativity higher than 1 was required. Higher cooperativity improved leaky expression tolerance, but only to a certain threshold. Higher leaky expression resulted in lower stable-state levels.<br />
</p><br />
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<br />
<br />
<br />
<h2><a name="model">The model</a></h2><br />
<p>The basis for the stochastic simulation of the mutual repressor switch was the following set of reactions that describe the dynamics of the switch:</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/22/Svn12_mrs_stoh1.PNG" /></p><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/b6/Svn12_mrs_stoh2.PNG" /></p><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/d0/Svn12_mrs_stoh3.PNG" /></p><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/da/Svn12_mrs_stoh4.PNG" /></p><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/19/Svn12_mrs_stoh5.PNG" /></p><br />
<p><img src="https://static.igem.org/mediawiki/2012/7/73/Svn12_mrs_stoh6.PNG" /></p><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/10/Svn12_mrs_stoh7.PNG" /></p><br />
<br />
<p><br />
Here:<br />
<ul style="margin-left:30px;"><br />
<li>Pro1 is construct 1 promoter (i.e. promoter 1 - constitutive);</li><br />
<li>Pro2 is construct 2 promoter (i.e. promoter 2 - constitutive);</li><br />
<li>Pro3 s construct 3 promoter (i.e. promoter 3 - constitutive);</li> <br />
<li>Pro4 is construct 4 promoter (i.e. promoter 4 - constitutive);</li> <br />
<li>Pro5 is construct 5 promoter (i.e. promoter 5 - constitutive);</li><br />
<li>Ind1 Is inducer 1 (i.e. signal 1), used to induce stable state 1;</li><br />
<li>Ind2 is inducer 2 (i.e. signal 2), used to induce stable state 2.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
[multi] is a variable, equal to a degree of oligomerization and is used to model cooperativity. Normal gene expression means constitutive gene expression, occuring when no repressor is bound.<br />
</p><br />
<br />
<br />
<p><br />
When inducer 1 is present, it binds PIP:KRAB and forms a complex, denoted as Ind1.PIP:KRAB. This complex is referred to as inactive PIP:KRAB, meaning PIP:KRAB that cannot bind to the promoter 3. When inducer 2 is present, it binds E:KRAB and forms a complex, denoted as Ind2.E:KRAB. This complex is referred to as inactive E:KRAB, meaning E:KRAB that cannot bind to the promoter 4.<br />
</p><br />
<br />
<br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
Simulation results are shown as concentrations of reporter for different states (BFP, mCitrine) indicating one of the two states as a function of time. No specific units were used, hence no absolute interpretation of the results' values in terms of units is in place. Switching between states was achieved using two signals (inducers) that were introduced into the system at appropriate time. The signals were modeled as a high number of molecules that triggered state induction and were removed at an appropriate time.<br />
</p><br />
<br />
<p><br />
Cooperativity was modeled as a degree of oligomerization, with repressor oligomers binding to a promoter.<br />
</p><br />
<br />
<p><br />
Protein production:degradation rate ratio was set to 10. Promoter unbinding (e.g. bound transcription factor unbinding from the target binding site) reactions were assumed to be 100 times slower than promoter binding reactions, which is in the range of typical transcription factors. Initial concentrations of proteins were 0 for all simulations.<br />
</p><br />
<br />
<p>Detailed parameter values for each simulation can be found in the corresponding simulation files that can be found <a href="https://2012.igem.org/Team:Slovenia/SourceCode">here</a>.</p><br />
<br />
<p><br />
The purpose of the first simulation was to show that the mutual repressor switch can exhibit bistability in a stochastic environment. No leaky expression was assumed, and no cooperativity (i.e. no TAL reprssor oligomerization). The following state-switching scenario was used in all stochastic simulations:<br />
<ul style="margin-left:30px;"><br />
<li>at time = 500, signal 2 was introduced, inducing stable state 2 (high mCitrine state); the signal gradually degraded, but stable state was preserved even after degradation;</li><br />
<li>at time = 2500, signal 1 was introduced to induce stable state 1 (high BFP state); the switch remained in a stable state after signal degradation;</li><br />
<li>at time = 4500, signal 2 was introduced again, switching the system back to stable state 2;at time = 6500, signal 1 was introduced again, switching the system to stable state 1.</li><br />
<li>at time = 6500, signal 1 was introduced again, switching the system to stable state 1.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
As shown in Figure 1, bistability was exhibited for this scenario, despite no cooperativity. However, as soon as sufficient leaky expression (e.g. 0.03 compared to protein production rate of 1) was introduced for each gene, bistability was lost (Figure 2).</p><br />
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<b>Figure 1.</b> Mutual repressor switch exhibiting transitions between stable states. No cooperativity and zero leaky expression were assumed here.<br />
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<b>Figure 2.</b> Mutual repressor switch exhibited no bistability when no cooperativity was used together with a relatively low leaky expression of 0.03.<br />
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<p><br />
For the switch to exhibit bistability in the presence of leaky expression, higher cooperativity values were required. Figure 3 shows bistable behavior of the switch for the cooperativity of 3 in the presence of leaky expression. Higher cooperativity led to higher leaky expression tolerance, but only to a certain leaky expression threshold, depending on other parameter values, such as production and degradation rates. Higher leaky expression also lowered the reached stable-state levels. The threshold for maximal cooperativity allowing bistability depended, just like leaky expression threshold, on other parameter values, such as production and degradation rates. Figure 4 shows that for very high cooperativity of 10, no bistability was observed at leaky expression rate of 0.05. <br />
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<b>Figure 3.</b> Bistability was obtained for cooperativity equal to 3 and leaky expression equal to 0.05.<br />
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<b>Figure 4.</b> Bistability was lost for cooperativity equal to 10 and leaky expression equal to 0.05.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitchCSim'>C#Sim model of the mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/SourceCode
Team:Slovenia/SourceCode
2012-10-26T21:11:37Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<!-- end main menu --><br />
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<br />
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<br/><br />
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<div id="dummy" style="background-color:transparent; position:absolute; left:870px; top:25px; width:115px; height:80px; z-index:100; opacity:0.0;"><br />
</div><br />
</a><br />
<br />
<br />
<h1>Source code</h1><br />
<br />
<h3>MATLAB - deterministic models</h3><br />
<p><br />
MATLAB source code of our deterministic models. Requires <a href="http://www.mathworks.com/matlabcentral/fileexchange/23629">export_fig</a> to work.<br />
<br/><a href="https://static.igem.org/mediawiki/2012/7/7f/MATLAB_deterministic_models_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>SGN Sim - stochastic models</h3><br />
<p><br />
<a href="http://www.cs.tut.fi/~sanchesr/SGN/SGNSim.html">SGN Sim</a> files with our stochastic model.<br/><br />
<a href="https://static.igem.org/mediawiki/2012/3/30/SGNSIM_stochastic_models_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>C#Sim</h3><br />
<p><br />
Executable (Windows), C# source code (with implementation of the mutual repressor switch and the positive feedback loop switch) and Visual Studio 2010 project files.<br />
<br/><a href="https://static.igem.org/mediawiki/2012/2/24/CSimIGEM_src.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>Pharmacokinetic real-time 3D simulation</h3><br />
<p><br />
Executables (Windows) and C++ source code for our pharmacokinetic 3D visualuzation. Requires <a href="http://freeglut.sourceforge.net/">freeglut library</a>. Hosted remotely due to large file size.<br/><br />
<a href="http://igem2012.fri.uni-lj.si/PK3D_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<br />
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</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/SourceCode
Team:Slovenia/SourceCode
2012-10-26T21:11:02Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<!-- end main menu --><br />
<br />
<br />
</div> <!-- end menu --><br />
<br />
<div id="main"><br />
<br/><br />
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</div><br />
</a><br />
<br />
<br />
<h1>Source code</h1><br />
<br />
<h3>MATLAB - deterministic models</h3><br />
<p><br />
MATLAB source code of our deterministic models. Requires <a href="http://www.mathworks.com/matlabcentral/fileexchange/23629">export_fig</a> to work.<br />
<br/><a href="https://static.igem.org/mediawiki/2012/7/7f/MATLAB_deterministic_models_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>SGN Sim - stochastic models</h3><br />
<p><br />
<a href="http://www.cs.tut.fi/~sanchesr/SGN/SGNSim.html">SGN Sim</a> files with our stochastic model.<br/><br />
<a href="https://static.igem.org/mediawiki/2012/3/30/SGNSIM_stochastic_models_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>C#Sim</h3><br />
<p><br />
Executable (Windows), C# source code (with implementation of the mutual repressor switch and the positive feedback loop switch) and Visual Studio 2010 project files.<br />
<br/><a href="https://static.igem.org/mediawiki/2012/2/24/CSimIGEM_src.zip">DOWNLOAD</a><br />
</p><br />
<br />
<h3>Pharmacokinetic real-time 3D simulation</h3><br />
<p><br />
Executables (Windows) and C++ source code for our pharmacokinetic 3D visualuzation. Requires <a href="http://freeglut.sourceforge.net/">freeglut library</a>. Hosted remotely due to large file size.<br/><br />
<a href="http://igem2012.fri.uni-lj.si/PK3D_Slovenia.zip">DOWNLOAD</a><br />
</p><br />
<br />
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</div><br />
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</body><br />
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</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/ModelingDerivation
Team:Slovenia/ModelingDerivation
2012-10-26T21:03:42Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<br />
<br />
<br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br />
<h1>Deterministic model derivation - genetic switch</h1><br />
<p><br />
The deterministic model for the mutual repressor switch was derived in the following way.<br />
</p><p><br />
We assumed the following binding site states were possible:<br />
<p><img src="https://static.igem.org/mediawiki/2012/2/2c/Svn12_model_derivation1.PNG"/></p><br />
</p><br />
<br />
<p>Active promoter state is a state leading to gene expression.<br />
</p><br />
<br />
<p>Construct 1 promoter state transitioning is described by a reaction:<img src="https://static.igem.org/mediawiki/2012/f/ff/Svn12_derivation1_eq1.png"/><br />
</p><br />
<br />
<p>Assuming equilibrium of binding and unbinding, we can write:<br />
<img src="https://static.igem.org/mediawiki/2012/f/f7/Svn12_derivation1_eq2.png"/><br />
</p><br />
<br />
<p><br />
The fractional occupancy of construct 1 promoter, f<sub>1</sub>, can be expressed as a ratio of active states to all states:<br />
<img src="https://static.igem.org/mediawiki/2012/b/b7/Svn12_derivation1_eq3.png" /><br />
</p><br />
<br />
<p><br />
The equations for other constructs take the same form.<br />
</p><br />
<br />
<p><br />
It follows that the fractional occupancy for construct 1 promoter is:<br />
<img src="https://static.igem.org/mediawiki/2012/b/ba/Svn12_derivation1_eq4.png" /><br />
</p><br />
<br />
<p>To account for non-linearity, an exponent n1 was added, and the equation generalized to:<br />
<img src="https://static.igem.org/mediawiki/2012/a/a9/Svn12_derivation1_eq5.png" /><br />
</p><br />
<br />
<br />
<p><br />
K<sub>r</sub> is the amount of TAL-A:KRAB it takes for f<sub>1</sub> to be equal to 50%.<br />
</p><br />
<br />
<p><br />
Derivation for constructs 2, 3 and 4 was similar except for different transcription factor names.<br />
</p><br />
<br />
<p><br />
Fractional occupancies were then used to construct a set of ordinary differential equations representing each protein production. Because each protein can be produced from different constructs, production rates (including leaky rates) were summed together. E.g., because TAL-B:KRAB is produced from both constructs 1 and 3, fractional occupancies f<sub>1</sub> and f<sub>3</sub> were used and corresponding terms summed to obtain:<br/><br/><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Svn12_mrs_det_d3_talbkrab.png" /><br />
</p><br />
<br />
<p><br />
Since construct 5 promoter has no binding sites and is active at all times, fractional occupancy of the promoter is equal to 1.<br />
</p><br />
<br />
<br />
<p><br />
The positive feedback loop switch model was derived in a similar manner.<br />
</p><br />
<br />
<br />
<br />
<br />
<hr><br />
<b><br />
<a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'>Deterministic model of the mutual repressor switch</a><br/><br />
<a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'>Deterministic model of the positive feedback loop switch</a><br />
<br />
</b><br />
<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitch
Team:Slovenia/TheSwitch
2012-10-26T20:14:44Z
<p>Dusanv: </p>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<h1>Overview</h1><br />
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<p><b>Introduction of complex regulatory circuits in mammalian cells requires the availability of a large number of designable orthogonal switches.<br />
We provide this new type of bistable toggle switch based on designable TAL regulators as a powerful tool to the synthetic biology community.</b></p><br />
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<ul style="padding-left:30px;"><br />
<li>We designed and tested several <b>TAL repressors, activators and their reporter plasmids</b> with their corresponding DNA-binding sites and promoters. The TAL repressors exhibited <b>over 90% repression</b> and TAL activators <b>enhanced expression over 1500 fold. </b> </li><br />
<li>We tested five induction systems for mammalian cells and adapted three of them for driving TAL regulators in genetic switches. </li><br />
<li>We designed and modeled a mutual repressor toggle switch based on two TAL repressors repressing each other. </li><br />
<li>Experimental results demonstrated <b>low stability</b> of the basic mutual repressor switch, based on the classical toggle switch topology. This was confirmed by modeling analysis, which indicated <b>high sensitivity to leaky gene expression and a requirement for high cooperativity</b> of repressors in order to exhibit bistable behavior. </li><br />
<li>We designed an <b>improved toggle switch that included additional positive feedback loops</b> based on orthogonal TAL repressors and activators competing for the same operator. </li><br />
<li>Modeling demonstrated <b>higher robustness in comparison to the mutual repressor switch</b> and <b>bistability even without cooperative binding to DNA</b>, since the positive feedback loop introduces the nonlinearity. </li><br />
<li>Experimental results of the improved switch demonstrated <b>two stable states at induction with corresponding inducers and a clear bimodal cell population distribution. </b> The switch remained in a stable state after removal of inducers, which confirmed the epigenetic bistability of our system. We included <b>numerous control experiments</b> demonstrating that the resultsare indeed due to the functional switch. </li><br />
<li><b><font color="red">(NEW)</font></b> Addition of the second inducer after the system has already adopted one state resulted in a <b>successful switch from one state into the other</b>, confirmed by three different experimental techniques . </li><br />
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<p style="padding-left:180px;"><b >Figure 1. Schematic representation of a positive feedback loop switch.</b></p><br />
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<table class="summary"><br />
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<p>Our project aims to <b>produce different biopharmaceuticals by engineered cells. </b> With tight but versatile regulation and safety in mind, we set out to contribute some fundamental advances to synthetic biology. </p><br />
<br />
<p>We considered two options of regulating production of therapeutic proteins in mammalian cells: </p><br />
<br />
<ul style="padding-left:30px;"><br />
<br />
<li>The first option could be a <b>prosthetic network</b> where the cells are engineered to <b>sense endogenous signals</b> such as for example blood glucose levels. This signal would control the production of a therapeutic protein (such as insulin), which in turn would affect the level of the endogenous signal (i.e. glucose) and be thus regulated by a kind of a feedback loop. This type of system can replace the function of a defective tissue or organ, hence the name prosthetic. Although a very attractive option, such a system would need to be tailor-made for each disease in particular. </li><br />
<li>For the second option we regarded a <b>cellular genetic network</b> that produces the therapeutic protein of choice, but could be <b>controlled by an external signal</b>, such as small molecular drugs, light or metabolites, which can be administered orally or topically. Instead of requiring a continuous presence of an activating or repressing signal, the system should rather function as a bistable or multistable switch, requiring only a signal pulse to change into any selected state and remain stable in this state. This type of toggle switch has already been implemented in mammalian cells based on prokaryotic transcription factors fused to the eukaryotic transactivator (e.g. VP16, VP64) or transrepressor (e.g. KRAB) domains (Karlsson et al., 2012). </li><br />
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<p>We consulted several physicians concerning different potential therapeutic applications of thistype of delivery system. Physicians by and large preferred the ability of the device to be controlled by the therapist to the autonomous switching by sensing internal physiological signals. An additional advantage of a controlled switch is that a very similar device core could be used for many different therapeutic applications.As examples of medical problems from the expertise of consulted physicians we considered wound healing, <a href = "https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC">hepatitis C infection</a> and <a href="https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease">cardiovascular ischemia</a>.For hepatitis C and ischaemic heart disease we constructed two sets of therapeuticeffectors and tested some of these effectors’ biologic activity. </p><br />
<p>Synthetic biology has already demonstrated the ability of a genetic device to assume different stable states. However, while prokaryotic regulators appear to be an efficient tool for device construction, their number is limited and currently does not allow the scalability required for construction of complex logical functions. On the other hand, modular DNA-binding protein domains such as zinc finger proteins or Transcription-activator like (TAL) domains can be designed to bind to almost any DNA sequence, providing high orthogonality. </p><br />
<p>Therefore the <b>challenge was to prepare an epigenetic switch based on designable DNA binding domains</b>, such as zinc finger proteins or TAL effectors that can be designed to recognize selected exogenous DNA sequences. This type of orthogonal switch would allow the simultaneous introduction of several bistable or multistabletoggle switches into mammalian cells for advanced level of control in different applications. To our knowledge, a <b>designed modular DNA binding protein-based toggle switch has not been implemented experimentally so far</b>, neither in mammalian nor in bacterial cells. This endeavor has to be supported by mathematical simulationto evaluate which type of genetic switch topology has the best performance, is more robust and which parameters are most critical for its performance. </p><br />
<p>Epigenetic toggle switches havepreviously been constructed based onnatural bacterial transcription factors (Gardner et al., 2000). An epigenetic toggle switch is a genetic network, designed to memorize its state and maintain expression of a gene of choice even after the inducer has been removed (Cherry & Adler, 2000). Only when a second signal (inducer) is provided to the cells, the system will switch to a different state, shutting off expression of the first protein and/or initiating production of a second one. This type of toggle switch allows the system to assume two discrete states. For our therapeutic purposes this could mean switching the production of a single protein on and off or selecting between the production of two different protein effectors. Therefore a combination of several switches would permit selection of advanced therapeutic regimens. </p><br />
<p>If we want the switches to function independently of each other andisolated from other cellular processes, they need to be based on the orthologous DNA-binding domains. We therefore decided to construct the genetic core of our device from TAL effectors, whose code of DNA recognition has been deciphered recently (Boch et al., 2009). TAL effectors have a modular structure, with each module recognizing a single base pair. TAL effectors, fused to theFokI nuclease, were successfullyadopted as highly specific and easily customizable genome editing tools (Christian et al, 2010; Bogdanove et al.; 2011, Li et al., 2011). For synthetic biologists, TAL effectors represent perfect candidates for engineering artificial transcription factors due to their high specificity, modularity, ability to target virtually any sequence and last, but not least, they do not exhibit toxicity when expressed in cells, except in case they target an essential endogenous gene. Indeed, in addition to endonucleases, transcription factors employing TALs as DNA binding domains have also been reported recently: transcription activators were constructed by fusing TALs to the mammalian activation domain VP16 or VP64 (Zhang et al., 2011) and, when our project already started, transcription repressors were reported, based on fusion of TALs with theKRAB heterochromatin silencing domain (Garg et al., 2012; Cong et al., 2012). </p><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., and Bonas, U. (2009) Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. <i>Science</i>. <b>326</b>, 1509-1512.<br/><br/><br />
Bogdanove, A. J., and Voytas, D. F. (2011) TAL Effectors: Customizable Proteins for DNA Targeting. <i>Science</i>. <b>333</b>, 1843-1846.<br/><br/><br />
Cherry, J. L. and Adler, F. R. (2000) How to make a Biological Switch. <i>J. Theor. Biol.</i> <b>203</b>, 117-133.<br/><br/><br />
Christian, M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., hummel, A., Bognadove, A. J., and Voytas, D. F. (2010) Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. <i>Genetics</i> <b>186</b>, 757-761.<br/><br/><br />
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F. (2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. <i>Nature</i>. <b>403</b>, 339-342.<br/><br/><br />
Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br/><br/><br />
Karlsson, M., Weber, W., and Fussenegger, M. (2012) Design and construction of synthetic gene networks in mammalian cells. <i>Methods Mol. Biol.</i> <b>813</b>, 359-376.<br/><br/><br />
Li, T., Huang, S., Jiang, W. Z., Wright, D., Spalding, M. H., Weeks, D., P., and Yang, B. (2011) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. <i>Nucleic Acids Res.</i> <b>39</b>, 359-372.<br/><br/><br />
Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'>Designed TAL regulators >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T20:14:21Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p><b><font color="red">(NEW)</font></b> Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href="https://2012.igem.org/Team:Slovenia/Parts#TALs">TAL regulators</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005">TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href="https://2012.igem.org/Team:Slovenia/Parts#reporters">reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href="http://partsregistry.org/Part:BBa_K323214">NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href="http://partsregistry.org/Part:BBa_K782007">NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<h3>Designed repressors</h3><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009">KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011">NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<h3><b><font color="red">(NEW)</font></b> TAL regulators are functional and nontoxic for multicellular animal</h3><br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrine under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter. It is exciting to see fluorescent tadpoles that were transfected with functional TAL activator and a reporter, which demonstrates that TAL activator is not deleterious for the development of the animal.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed 1 and 3 days after introduction of plasmids under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results obtained in collaboration with <a href="https://2012.igem.org/Team:Evry">Evry team</a> (thank you froggies) thus demonstrate that TAL-based logic can be used also in the whole animal, which could be used as an excellent model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchControls
Team:Slovenia/TheSwitchControls
2012-10-26T19:55:12Z
<p>Dusanv: </p>
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</head><br />
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<body><br />
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<a style="position:absolute; top:0px; left:490px;" href="https://2012.igem.org/Main_Page"><b></b></a><br />
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<!--<div id="container">--><br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<!-- end main menu --><br />
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</div> <!-- end menu --><br />
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</a><br />
<br />
<br/><br />
<h2>Controls</h2><br />
<br />
<br />
<br />
<!-- 1 --><br />
<h3>Inducible TAL regulators</h3><br />
<hr color="gray"/><br />
<br />
<br/><br />
<p><br />
We improved the induction system we <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch">previously used</a> by placing the binding sites upstream of the promoter and by fusing CL1-PEST degradation tags to TAL effectors. Before using them in our TAL-based switches we had to determine their functionality. To test them we cotransfected inducible repressors with a reporter and observed the repression with and without the addition of the appropriate inducer. The tests show strong repression of the reporter upon induction, confirming that the inducible TAL repressor is expressed and functions as expected.<br />
</p><br />
<br />
<br />
<table width="80%" align="center"><br />
<tr ><br />
<td style="padding-right:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/e/e9/Svn12_ctrl_1a.png<br />
"></td><br />
<td style="padding-left:10px;" ><img width="100%" src="https://static.igem.org/mediawiki/2012/8/87/Svn12_ctrl_1b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td style="padding-right:10px;"><p><b>Erythromycin induces the expression of a TAL repressor, which represses a luciferase reporter.<br />
</b><br />
HEK293T cells were cotransfected with transfection control (10 ng Renilla luciferase), [AB]_PCMV_fLuc reporter and ETR_PCMV_PEST-CL1-TALA:KRAB (both 10 ng) and E:KRAB (100 ng). Two hours post-transfection the cells were stimulated with 2 µg/ml erythromycin.<br />
</p></td><br />
<td style="padding-left:10px;"><p><b>Pristinamycin induces the expression of a TAL repressor, which represses a luciferase reporter.<br />
</b><br />
HEK293T cells were cotransfected with transfection control (10 ng Renilla luciferase), [AB]_PCMV_fLuc reporter and PIR_PCMV_PEST-CL1-TALA:KRAB (borh 10 ng) and PIP:KRAB (100 ng). Two hours post-transfection the cells were stimulated with 2 µg/ml pristinamycin.<br />
</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<!-- 2 --><br />
<br/><br />
<br/><br />
<h3>Specificity of TAL effectors </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>For the switch to function properly, all of TAL effectors need to exhibit high specificity and orthogonality. We tested different combinations of TAL operons and TAL activators (Figure) to control for any cross-reactivity and binding specificity. <br />
When a reporter under the TAL operon was cotransfected with its matching TAL activator, fluorescence was detected. When the transfected reported plasmid‘s operon did not match the cotransfected TAL activator, no flourescence was detected even if it contained 10 copies of two other TAL binding sites. This demonstrates that TAL regulators bind and exert activity specifically at their binding sites and <b>high degree of orthogonality. </b><br />
</p><br />
<table width="70%" align="center"><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/b/be/Svn12_ctrl_2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
<p><b>TAL activator specifically recognizes its corresponding DNA-binding site. </b><br />
HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 (a,b) or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).<br />
</p></td><br />
</tr><br />
</table><br />
<p>For the switch to function properly, all of TAL effectors need to exhibit high specificity and orthogonality. We tested different combinations of TAL operons and TAL activators (Figure) to control for any cross-reactivity and binding specificity. <br />
When a reporter under the TAL operon was cotransfected with its matching TAL activator, fluorescence was detected. When the transfected reported plasmid‘s operon did not match the cotransfected TAL activator, no flourescence was detected even if it contained 10 copies of two other TAL binding sites. This demonstrates that TAL regulators bind and exert activity specifically at their binding sites and <b>high degree of orthogonality. </b><br />
</p><br />
<br />
<br />
<!-- 3 --><br />
<br/><br />
<br/><br />
<h3>Test of minimal promoter leakage amplified by autoactivator </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>In order to investigate the leakage of minimal promoters in the switch, we performed two controls that included the plasmids of one or the other state of the switch but no induction system. <br />
We observed flourescence corresponding to the transfected plasmids in few cells, indicating some promoter leakage. Even though we determined a minimal leakage of the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers the positive feedback loop and causes further activation of the system and expression of the reporter. Leakage of the minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a repressor.<br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<td style="padding-right:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/4/46/Svn12_ctrl_3a1.png<br />
"></td><br />
<td style="padding-left:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/3/39/Svn12_ctrl_3b1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td style="padding-right:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/7/76/Svn12_ctrl_3a2.png<br />
"></td><br />
<td style="padding-left:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/0/0e/Svn12_ctrl_3b2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td style="padding-right:10px;"><br />
<p>[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP (both 20 ng) <br />
</p></td><br />
<td style="padding-left:10px;"><br />
<p>[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng) <br />
</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<!-- 4 --><br />
<br/><br />
<br/><br />
<h3>Test of bistability of the positive feedback loop </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>In order to investigate the leakage of minimal promoters in the switch, we performed two controls that included the plasmids of one or the other state of the switch but no induction system. <br />
We observed flourescence corresponding to the transfected plasmids in few cells, indicating some promoter leakage. Even though we determined a minimal leakage of the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers the positive feedback loop and causes further activation of the system and expression of the reporter. Leakage of the minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a repressor.<br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/3/3a/Svn12_ctrl_4a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/4/4b/Svn12_ctrl_4a2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng) <br />
</td><br />
</tr><br />
</table><br />
</td><br />
<td valign="top" style="padding-left:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/c/cc/Svn12_ctrl_4b.png<br />
"></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<!-- 5 --><br />
<br/><br />
<br/><br />
<h3>Illustration of the readout of results of the flow cytometry with transiently transfected mammalian cells that are able to produce two fluorescent reporters<br />
</h3><br />
<hr color="gray"/><br />
<table width="80%" align="center"><br />
<tr ><br />
<td width="50%" valign="middle" style="padding-right:10px; padding-top:40px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/23/Svn12_ctrl_5a.png<br />
"></td><br />
<td width="50%" valign="middle" style="padding-left:10px; " ><img width="100%" src="https://static.igem.org/mediawiki/2012/f/f1/Svn12_ctrl_5b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td width="50%" valign="top" style="padding-right:10px;"><p><b>Schematic representation of populations of cells transfected with the switch as measured by flow cytometry.</b> Upper left quadrant (Q1): cells producing only BFP; lower right quadrant (Q3): cells producing only mCitrine; right top quadrant (Q2): cells producing both BFP and mCitrine; lower left quadrant (Q4): nontransfected cells and cells that produce neither BFP nor mCitrine. The efficiency of mammalian cell transfection was typicaly between 30-50%.<br />
</p><br />
</p></td><br />
<td width="50%" valign="top" style="padding-left:30px;"><p><b>Nontransfected cells.</b><br />
</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<!-- 6 --><br />
<br/><br />
<br/><br />
<h3>Induction of state I <br />
</h3><br />
<hr color="gray"/><br />
<br/><br />
<p>In order to remotely control the switch by small moleclar weight inductors, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (zero-level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression). <br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/6/6c/Svn12_ctrl_6a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/8/87/Svn12_ctrl_6a2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), <br />
PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/9/92/Svn12_ctrl_6b.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16, (all 40ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 7 --><br />
<br/><br />
<br/><br />
<h3>Induction of state II<br />
</h3><br />
<hr color="gray"/><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/a/a3/Svn12_ctrl_7a.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/0/0e/Svn12_ctrl_7b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16 (all 5 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16 (both 10ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/4/48/Svn12_ctrl_7c.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, (all 40ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 8 --><br />
<br/><br />
<br/><br />
<h3>The switch coupled with both induction systems </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>Next we tested the response of the switch when all of the first level plasmids, but plasmids of only one of the induction systems are present. This should result in the constitutively active promoters on the first level plasmids without their corresponding repressors, and (in the absence of inducer) constitutively repressed promoters on the first level plasmids of the complete induction system. A missing repressor thus in effect simulates a constitutive presence of the corresponding inducer. As expected, the system behaves in the same way as when it is induced. Without the addition of PIP:KRAB, the system expresses BFP (state I) and without the addition of E:KRAB the system expresses mCitrine (state II).<br />
</p><br />
<br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="50%" valign="top"><br />
<br />
<br />
<table align="center"><br />
<tr width="100%"><br />
<td width="50%" valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/22/Svn12_ctrl_8a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/b/b0/Svn12_ctrl_8a2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
PCMV_PIP:KRAB (200ng),<br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, <br />
PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng),<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<br />
<br />
<br />
<td style="padding-left:10px;" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/2e/Svn12_ctrl_8b1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/23/Svn12_ctrl_8b2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
PCMV_E:KRAB (200 ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 10 --><br />
<br/><br />
<br/><br />
<h3>The switch coupled with both induction systems - NOT INDUCED <br />
<br />
</h3><br />
<hr color="gray"/><br />
<br/><br />
<p>To finally prove the bistability of the complete switch (including the complete induction systems) we transfected the cell with all of the plasmids and left them either unstimulated or stimulated them with both of the inducers at once. With microscopy and flow cytometry we observed two populations of cells both expressing only one of the fluorescent reporters, confirming our expectations of bistability as even in the absence of inducers or presence of both inducers<br />
the system stochastically switches to one or the other of the two clearly defined stable states. <br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/c/c3/Svn12_ctrl_10a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/5/52/Svn12_ctrl_10a2.png<br />
"></td><br />
</tr><br />
<br />
<br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/3/34/Svn12_ctrl_10b.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16 (all 40ng)<br />
PCMV_PIP:KRAB, PCMV_E:KRAB (both 80 ng).<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 11 --><br />
<br/><br />
<br/><br />
<h3>The switch coupled with both induction systems - both inducers present simultaneously </h3><br />
<hr color="gray"/><br />
<br/><p>Some of the judges at iGEM were curious what would happen if the system is stimulated with both inducers symultaneously. This type of combination of both signals results in the electronic circuits as an undefined state. Experimental results for our switch suggest that both reporters are effectively repressed, as we determined that transcription factor with KRAB is able to repress the effect of a transcription factor comprisong VP16 activator. <br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/0/0b/Svn12_ctrl_11a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/9/97/Svn12_ctrl_11a2.png<br />
"></td><br />
</tr><br />
<br />
<tr width="100%"><br />
<td><br />
[A]_PMIN_TALB:KRAB (10 ng), [A]_PMIN_TALA:VP16_t2A_BFP (2 ng), <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine (10 ng), [B]_PMIN_TALB:VP16, (2 ng), <br />
PCMV_[PIR]_TALB:KRAB (20 ng), PCMV_[PIR]_TALA:VP16 (5 ng),<br />
PCMV_[ETR]_TALA:KRAB (20 ng), PCMV_[ETR]_TALB:VP16, ( 5 ng),<br />
PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). <br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/9/93/Svn12_ctrl_11b.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16 (all 40ng)<br />
PCMV_PIP:KRAB, PCMV_E:KRAB (both 80 ng).<br />
<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 9 --><br />
<br/><br />
<br/><br />
<h3>Stimulation<br />
</h3><br />
<hr color="gray"/><br />
We prepared a switch with inducible systems comprising CL1-PEST degradation tags (see figure/control 1), which should allow faster switching between states. We tested how these improved induction systems induce one state of the switch or the other. We also tested if the switch retains the stable state if the inducer is removed.<br />
<br/><br />
<br/><br />
<table width="95%" align="center"><br />
<tr ><br />
<td width="50%" valign="middle" style="padding-right:10px; "><img width="100%" src="https://static.igem.org/mediawiki/2012/9/9f/Svn12_ctrl_9a.png<br />
"></td><br />
<td width="50%" valign="middle" style="padding-left:10px; " ><img width="100%" src="https://static.igem.org/mediawiki/2012/3/3c/Svn12_ctrl_9b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td width="50%" valign="top" style="padding-right:10px;"><b>Cells constantly stimulated with erythromycin (day 5 after the transfection).</b><br />
HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng),<br /> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng),<br /> [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng),<br /> [B]_PMIN_TALB:VP16 (7 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br /> PCMV_PIP:KRAB,<br /> PCMV_E:KRAB (both 300 ng). Erythromycin was added to final concentration of 2 µg/ml. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
<br />
</td><br />
<td width="50%" valign="top" style="padding-left:30px;"><b>Cells constantly stimulated with pristinamycin (day 5 after the transfection).</b><br />
HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng),<br /> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng),<br /> [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng),<br /> [B]_PMIN_TALB:VP16 (7 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br /> PCMV_PIP:KRAB,<br /> PCMV_E:KRAB (both 300 ng). Pristinamycin was added to final concentration of 2 µg/ml. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
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<td width="50%" valign="middle" style="padding-right:10px; "><img width="100%" src="https://static.igem.org/mediawiki/2012/a/aa/Svn12_ctrl_12a.png<br />
"></td><br />
<td width="50%" valign="middle" style="padding-left:10px; " ><img width="100%" src="https://static.igem.org/mediawiki/2012/a/a0/Svn12_ctrl_12b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td width="50%" valign="top" style="padding-right:10px;"><b>After removal of erythromycin the switch stably expresses mCitrine</b> HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), <br/> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng), <br/>[B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng), <br/>[B]_PMIN_TALB:VP16 (7 ng), <br/>[PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng), <br/>[PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng), <br/> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng), <br/> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br/> PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng). <br/>Erythromycin was added to final concentration of 2 µg/ml. After one day the cell medium was changed, removing the erythromycin. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
</td><br />
<td width="50%" valign="top" style="padding-left:30px;"><b>After removal of pristinamycin the switch stably expresses mCitrine</b><br />
HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), <br/> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng),<br/> [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng),<br/> [B]_PMIN_TALB:VP16 (7 ng), <br/> [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng),<br/> [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng),<br/> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng),<br/> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br/> PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng).<br/> Pristinamycin was added to final concentration of 2 µg/ml. After one day the cell medium was changed, removing the pristinamycin. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
</td><br />
</tr><br />
</table><br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'>Safety mechanisms >></a><br />
</b><br />
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</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchControls
Team:Slovenia/TheSwitchControls
2012-10-26T19:54:46Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
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<a href="https://2012.igem.org/Main_Page"><br />
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</div><br />
</a><br />
<br />
<br/><br />
<h2>Controls</h2><br />
<br />
<br />
<br />
<!-- 1 --><br />
<br/><br />
<br/><br />
<h3>Inducible TAL regulators</h3><br />
<hr color="gray"/><br />
<br />
<br/><br />
<p><br />
We improved the induction system we <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch">previously used</a> by placing the binding sites upstream of the promoter and by fusing CL1-PEST degradation tags to TAL effectors. Before using them in our TAL-based switches we had to determine their functionality. To test them we cotransfected inducible repressors with a reporter and observed the repression with and without the addition of the appropriate inducer. The tests show strong repression of the reporter upon induction, confirming that the inducible TAL repressor is expressed and functions as expected.<br />
</p><br />
<br />
<br />
<table width="80%" align="center"><br />
<tr ><br />
<td style="padding-right:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/e/e9/Svn12_ctrl_1a.png<br />
"></td><br />
<td style="padding-left:10px;" ><img width="100%" src="https://static.igem.org/mediawiki/2012/8/87/Svn12_ctrl_1b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td style="padding-right:10px;"><p><b>Erythromycin induces the expression of a TAL repressor, which represses a luciferase reporter.<br />
</b><br />
HEK293T cells were cotransfected with transfection control (10 ng Renilla luciferase), [AB]_PCMV_fLuc reporter and ETR_PCMV_PEST-CL1-TALA:KRAB (both 10 ng) and E:KRAB (100 ng). Two hours post-transfection the cells were stimulated with 2 µg/ml erythromycin.<br />
</p></td><br />
<td style="padding-left:10px;"><p><b>Pristinamycin induces the expression of a TAL repressor, which represses a luciferase reporter.<br />
</b><br />
HEK293T cells were cotransfected with transfection control (10 ng Renilla luciferase), [AB]_PCMV_fLuc reporter and PIR_PCMV_PEST-CL1-TALA:KRAB (borh 10 ng) and PIP:KRAB (100 ng). Two hours post-transfection the cells were stimulated with 2 µg/ml pristinamycin.<br />
</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<!-- 2 --><br />
<br/><br />
<br/><br />
<h3>Specificity of TAL effectors </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>For the switch to function properly, all of TAL effectors need to exhibit high specificity and orthogonality. We tested different combinations of TAL operons and TAL activators (Figure) to control for any cross-reactivity and binding specificity. <br />
When a reporter under the TAL operon was cotransfected with its matching TAL activator, fluorescence was detected. When the transfected reported plasmid‘s operon did not match the cotransfected TAL activator, no flourescence was detected even if it contained 10 copies of two other TAL binding sites. This demonstrates that TAL regulators bind and exert activity specifically at their binding sites and <b>high degree of orthogonality. </b><br />
</p><br />
<table width="70%" align="center"><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/b/be/Svn12_ctrl_2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
<p><b>TAL activator specifically recognizes its corresponding DNA-binding site. </b><br />
HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 (a,b) or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).<br />
</p></td><br />
</tr><br />
</table><br />
<p>For the switch to function properly, all of TAL effectors need to exhibit high specificity and orthogonality. We tested different combinations of TAL operons and TAL activators (Figure) to control for any cross-reactivity and binding specificity. <br />
When a reporter under the TAL operon was cotransfected with its matching TAL activator, fluorescence was detected. When the transfected reported plasmid‘s operon did not match the cotransfected TAL activator, no flourescence was detected even if it contained 10 copies of two other TAL binding sites. This demonstrates that TAL regulators bind and exert activity specifically at their binding sites and <b>high degree of orthogonality. </b><br />
</p><br />
<br />
<br />
<!-- 3 --><br />
<br/><br />
<br/><br />
<h3>Test of minimal promoter leakage amplified by autoactivator </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>In order to investigate the leakage of minimal promoters in the switch, we performed two controls that included the plasmids of one or the other state of the switch but no induction system. <br />
We observed flourescence corresponding to the transfected plasmids in few cells, indicating some promoter leakage. Even though we determined a minimal leakage of the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers the positive feedback loop and causes further activation of the system and expression of the reporter. Leakage of the minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a repressor.<br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<td style="padding-right:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/4/46/Svn12_ctrl_3a1.png<br />
"></td><br />
<td style="padding-left:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/3/39/Svn12_ctrl_3b1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td style="padding-right:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/7/76/Svn12_ctrl_3a2.png<br />
"></td><br />
<td style="padding-left:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/0/0e/Svn12_ctrl_3b2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td style="padding-right:10px;"><br />
<p>[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP (both 20 ng) <br />
</p></td><br />
<td style="padding-left:10px;"><br />
<p>[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng) <br />
</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<!-- 4 --><br />
<br/><br />
<br/><br />
<h3>Test of bistability of the positive feedback loop </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>In order to investigate the leakage of minimal promoters in the switch, we performed two controls that included the plasmids of one or the other state of the switch but no induction system. <br />
We observed flourescence corresponding to the transfected plasmids in few cells, indicating some promoter leakage. Even though we determined a minimal leakage of the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers the positive feedback loop and causes further activation of the system and expression of the reporter. Leakage of the minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a repressor.<br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/3/3a/Svn12_ctrl_4a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/4/4b/Svn12_ctrl_4a2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng) <br />
</td><br />
</tr><br />
</table><br />
</td><br />
<td valign="top" style="padding-left:10px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/c/cc/Svn12_ctrl_4b.png<br />
"></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<!-- 5 --><br />
<br/><br />
<br/><br />
<h3>Illustration of the readout of results of the flow cytometry with transiently transfected mammalian cells that are able to produce two fluorescent reporters<br />
</h3><br />
<hr color="gray"/><br />
<table width="80%" align="center"><br />
<tr ><br />
<td width="50%" valign="middle" style="padding-right:10px; padding-top:40px;"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/23/Svn12_ctrl_5a.png<br />
"></td><br />
<td width="50%" valign="middle" style="padding-left:10px; " ><img width="100%" src="https://static.igem.org/mediawiki/2012/f/f1/Svn12_ctrl_5b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td width="50%" valign="top" style="padding-right:10px;"><p><b>Schematic representation of populations of cells transfected with the switch as measured by flow cytometry.</b> Upper left quadrant (Q1): cells producing only BFP; lower right quadrant (Q3): cells producing only mCitrine; right top quadrant (Q2): cells producing both BFP and mCitrine; lower left quadrant (Q4): nontransfected cells and cells that produce neither BFP nor mCitrine. The efficiency of mammalian cell transfection was typicaly between 30-50%.<br />
</p><br />
</p></td><br />
<td width="50%" valign="top" style="padding-left:30px;"><p><b>Nontransfected cells.</b><br />
</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<!-- 6 --><br />
<br/><br />
<br/><br />
<h3>Induction of state I <br />
</h3><br />
<hr color="gray"/><br />
<br/><br />
<p>In order to remotely control the switch by small moleclar weight inductors, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (zero-level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression). <br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/6/6c/Svn12_ctrl_6a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/8/87/Svn12_ctrl_6a2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), <br />
PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/9/92/Svn12_ctrl_6b.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16, (all 40ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 7 --><br />
<br/><br />
<br/><br />
<h3>Induction of state II<br />
</h3><br />
<hr color="gray"/><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/a/a3/Svn12_ctrl_7a.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/0/0e/Svn12_ctrl_7b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16 (all 5 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16 (both 10ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/4/48/Svn12_ctrl_7c.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, (all 40ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 8 --><br />
<br/><br />
<br/><br />
<h3>The switch coupled with both induction systems </h3><br />
<hr color="gray"/><br />
<br/><br />
<p>Next we tested the response of the switch when all of the first level plasmids, but plasmids of only one of the induction systems are present. This should result in the constitutively active promoters on the first level plasmids without their corresponding repressors, and (in the absence of inducer) constitutively repressed promoters on the first level plasmids of the complete induction system. A missing repressor thus in effect simulates a constitutive presence of the corresponding inducer. As expected, the system behaves in the same way as when it is induced. Without the addition of PIP:KRAB, the system expresses BFP (state I) and without the addition of E:KRAB the system expresses mCitrine (state II).<br />
</p><br />
<br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="50%" valign="top"><br />
<br />
<br />
<table align="center"><br />
<tr width="100%"><br />
<td width="50%" valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/22/Svn12_ctrl_8a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/b/b0/Svn12_ctrl_8a2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
PCMV_PIP:KRAB (200ng),<br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, <br />
PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng),<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<br />
<br />
<br />
<td style="padding-left:10px;" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/2e/Svn12_ctrl_8b1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/2/23/Svn12_ctrl_8b2.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td ><br />
PCMV_E:KRAB (200 ng)<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 10 --><br />
<br/><br />
<br/><br />
<h3>The switch coupled with both induction systems - NOT INDUCED <br />
<br />
</h3><br />
<hr color="gray"/><br />
<br/><br />
<p>To finally prove the bistability of the complete switch (including the complete induction systems) we transfected the cell with all of the plasmids and left them either unstimulated or stimulated them with both of the inducers at once. With microscopy and flow cytometry we observed two populations of cells both expressing only one of the fluorescent reporters, confirming our expectations of bistability as even in the absence of inducers or presence of both inducers<br />
the system stochastically switches to one or the other of the two clearly defined stable states. <br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/c/c3/Svn12_ctrl_10a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/5/52/Svn12_ctrl_10a2.png<br />
"></td><br />
</tr><br />
<br />
<br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/3/34/Svn12_ctrl_10b.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16 (all 40ng)<br />
PCMV_PIP:KRAB, PCMV_E:KRAB (both 80 ng).<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 11 --><br />
<br/><br />
<br/><br />
<h3>The switch coupled with both induction systems - both inducers present simultaneously </h3><br />
<hr color="gray"/><br />
<br/><p>Some of the judges at iGEM were curious what would happen if the system is stimulated with both inducers symultaneously. This type of combination of both signals results in the electronic circuits as an undefined state. Experimental results for our switch suggest that both reporters are effectively repressed, as we determined that transcription factor with KRAB is able to repress the effect of a transcription factor comprisong VP16 activator. <br />
</p><br />
<table width="95%" align="center"><br />
<tr width="100%"><br />
<br />
<br />
<td style="padding-right:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/0/0b/Svn12_ctrl_11a1.png<br />
"></td><br />
</tr><br />
<tr width="100%"><br />
<td ><img width="100%" src="https://static.igem.org/mediawiki/2012/9/97/Svn12_ctrl_11a2.png<br />
"></td><br />
</tr><br />
<br />
<tr width="100%"><br />
<td><br />
[A]_PMIN_TALB:KRAB (10 ng), [A]_PMIN_TALA:VP16_t2A_BFP (2 ng), <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine (10 ng), [B]_PMIN_TALB:VP16, (2 ng), <br />
PCMV_[PIR]_TALB:KRAB (20 ng), PCMV_[PIR]_TALA:VP16 (5 ng),<br />
PCMV_[ETR]_TALA:KRAB (20 ng), PCMV_[ETR]_TALB:VP16, ( 5 ng),<br />
PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). <br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
<br />
<br />
<td style="padding-left:10px;" width="60%" valign="top"><br />
<table width="100%" align="center"><br />
<tr width="100%"><br />
<td valign="top"><img width="100%" src="https://static.igem.org/mediawiki/2012/9/93/Svn12_ctrl_11b.png<br />
"></td><br />
</tr><br />
<br />
<tr ><br />
<td ><br />
[A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 20 ng), <br />
PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[TRE]_TALA:KRAB, PCMV_[TRE]_TALB:VP16 (all 40ng)<br />
PCMV_PIP:KRAB, PCMV_E:KRAB (both 80 ng).<br />
<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<br />
<br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- 9 --><br />
<br/><br />
<br/><br />
<h3>Stimulation<br />
</h3><br />
<hr color="gray"/><br />
We prepared a switch with inducible systems comprising CL1-PEST degradation tags (see figure/control 1), which should allow faster switching between states. We tested how these improved induction systems induce one state of the switch or the other. We also tested if the switch retains the stable state if the inducer is removed.<br />
<br/><br />
<br/><br />
<table width="95%" align="center"><br />
<tr ><br />
<td width="50%" valign="middle" style="padding-right:10px; "><img width="100%" src="https://static.igem.org/mediawiki/2012/9/9f/Svn12_ctrl_9a.png<br />
"></td><br />
<td width="50%" valign="middle" style="padding-left:10px; " ><img width="100%" src="https://static.igem.org/mediawiki/2012/3/3c/Svn12_ctrl_9b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td width="50%" valign="top" style="padding-right:10px;"><b>Cells constantly stimulated with erythromycin (day 5 after the transfection).</b><br />
HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng),<br /> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng),<br /> [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng),<br /> [B]_PMIN_TALB:VP16 (7 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br /> PCMV_PIP:KRAB,<br /> PCMV_E:KRAB (both 300 ng). Erythromycin was added to final concentration of 2 µg/ml. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
<br />
</td><br />
<td width="50%" valign="top" style="padding-left:30px;"><b>Cells constantly stimulated with pristinamycin (day 5 after the transfection).</b><br />
HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng),<br /> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng),<br /> [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng),<br /> [B]_PMIN_TALB:VP16 (7 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng),<br /> [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng),<br /> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br /> PCMV_PIP:KRAB,<br /> PCMV_E:KRAB (both 300 ng). Pristinamycin was added to final concentration of 2 µg/ml. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<!-- 12 --><br />
<br/><br />
<br/><br />
<h3>Inducer removal <br />
</h3><br />
<hr color="gray"/><br />
<table width="95%" align="center"><br />
<tr ><br />
<td width="50%" valign="middle" style="padding-right:10px; "><img width="100%" src="https://static.igem.org/mediawiki/2012/a/aa/Svn12_ctrl_12a.png<br />
"></td><br />
<td width="50%" valign="middle" style="padding-left:10px; " ><img width="100%" src="https://static.igem.org/mediawiki/2012/a/a0/Svn12_ctrl_12b.png<br />
"></td><br />
</tr><br />
<tr ><br />
<td width="50%" valign="top" style="padding-right:10px;"><b>After removal of erythromycin the switch stably expresses mCitrine</b> HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), <br/> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng), <br/>[B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng), <br/>[B]_PMIN_TALB:VP16 (7 ng), <br/>[PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng), <br/>[PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng), <br/> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng), <br/> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br/> PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng). <br/>Erythromycin was added to final concentration of 2 µg/ml. After one day the cell medium was changed, removing the erythromycin. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
</td><br />
<td width="50%" valign="top" style="padding-left:30px;"><b>After removal of pristinamycin the switch stably expresses mCitrine</b><br />
HEK293T cells were cotransfected with the following plasmids: [A]_PMIN_TALB:KRAB (21 ng), <br/> [A]_PMIN_TALA:VP16_t2A_BFP (7 ng),<br/> [B]_PMIN_TALA:KRAB_t2A_mCitrine (21 ng),<br/> [B]_PMIN_TALB:VP16 (7 ng), <br/> [PIR]_PCMV_ CL1-PEST:TALB:KRAB (30 ng),<br/> [PIR]_PCMV_ CL1-PEST:TALA:VP16 (10 ng),<br/> [ETR]_PCMV_ CL1-PEST:TALA:KRAB (30 ng),<br/> [ETR]_PCMV_ CL1-PEST:TALB:VP16 (10 ng),<br/> PCMV_PIP:KRAB, PCMV_E:KRAB (both 300 ng).<br/> Pristinamycin was added to final concentration of 2 µg/ml. After one day the cell medium was changed, removing the pristinamycin. Fluorescence was measured by flow cytometry 5 days after transfection.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'>Safety mechanisms >></a><br />
</b><br />
<br />
</div><br />
<!--</div>--><br />
<br />
<br />
<br />
</body><br />
<br />
<br />
</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch
Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch
2012-10-26T19:52:20Z
<p>Dusanv: </p>
<hr />
<div><html><br />
<head><br />
<meta http-equiv="X-UA-Compatible" content="IE=edge" /><br />
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<style type="text/css"><br />
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*:first-child+html .clearfix{min-height:1%;}<br />
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img { display: block; margin-left: auto; margin-right: auto }<br />
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a:hover {border-bottom:1px solid #505050; color:#303030; text-decoration:none;}<br />
a img {border:0;}<br />
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<br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<h1> Positive feedback loop switch</h1><br />
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<p>We designed an upgraded <b>bistable genetic toggle switch based on orthogonal TAL repressors and activators</b> which is composed of a pair of mutual repressors and a pair of activators,upgradingthe classical toggle switch with positive feedback loop. </p><br />
<p>Simulations of the positive feedback loop switch demonstrated <b>bistability even at low or no cooperativity</b>. </p><br />
<p>Stochastic and deterministic simulations indicate <b>higher robustness</b> in comparison to the mutual repressor switch. </p> <br />
<p>We experimentally tested the switch by monitoring production of two fluorescent protein reporters, confirming a <b>clear bimodal distribution of reporter fluorescence</b> and demonstrated adoption of <b>stable states</b> by induction with corresponding inducer molecules. </p><br />
<p>The switch <b>persisted in a stable state</b> after the removal of inducer molecules, which confirmed the epigenetic bistability of our system. </p><br />
<p><b><font color="red">(NEW)</font></b> Cells could <b>switch from one state to the other</b> by the addition of the second inducer, which was confirmed by two methods. </p> <br />
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<h2>Bistable genetic switch based on non-cooperative elements</h2><br />
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<p>Mathematical analysis of genetic switches from the literature indicates that cooperativity, which introduces a nonlinear response, is required for a functional bistable switch, consisting of two mutual repressors (Cherry et al. 2000). Macia et al. (2009) and Widder et al. (2009) proposed that <b>bistability could be introduced by non-cooperative elements, when nonlinearity is introduced</b> for exampleif protein A is able to repress the transcription of protein B and at the same time activate its own transcription and <i>vice versa</i> (Figure 1). </p> <br />
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<td class="invisible" style="text-align:justify; width:70%;"> <b>Figure 1. Macia et al. (2009) and Widder et al. (2009) proposed a mathematical solution for creation of a bistable switch that would not require the cooperative binding of transcriptional regulators. </b> According to the proposed model, transcription factor A should activate its own transcription and repress the transcription of gene B and <i>vice versa.</i> <br />
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<p>We, as molecular biologists, werenot aware of a transcriptional effector or effector pair that could act simultaneously as a repressor and activator, therefore we were not surprised that, to our knowledge, this type of bistable switch has not yet been experimentally implemented. </p> <br />
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<p>The crucial moment in our project was when we realized that the ability to design <b>TAL repressors and activators directed against the same binding site could offer a solution to this problem and provide a unique opportunity to construct orthogonal bistable switches based on noncooperative elements (Figure 2). </b> </p><br />
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<td class="invisible" style="text-align:justify; width:100%;"> <b>Figure 2. A repressor (left) and an activator (right) competing for the same binding site. </b> <br />
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<h2>Results</h2><br />
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<h3>Design</h3><br />
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<p>We designed an <b>upgraded mutual repressor switch, </b> introducing <b>two additional positive feedback loops</b> (Figure 3), consisting of two TAL activators targeted against the same binding sites as apair of mutual TAL repressors. In other words, <b>rather than having the same protein function as an activator and repressor, we used an activator and repressor pair, competing for the same operator. </b> The same binding sequence for the activator and repressor introduced nonlinearity required for the bistability based oncompetition for the binding site. For the purpose of our project, <b>we designed a bistable switch with a positive feedback loop, capable of switching between the two states through regulation by inducer molecules</b> (Figure 4). </p><br />
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<b>Figure 3. Scheme of the genetic circuit for the bistable toggle switch composed of a pair of mutual repressors and a pair of autoactivators under the control of two operators. </b><br />
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<b>Figure 4. Designed states of the bistable toggle switch with a positive feedback loop. </b> (A) Addition of inducer 1 triggers dissociation of its cognate repressor from its DNA-binding site. As a consequence, repressor A and activator B are transcribed. Activator B further activates transcription of repressor A and itself, which forms the positive feedback loop of the switch state one. Meanwhile repressor A inhibits the other state of the switch. (B) When the inducer is removed, the inducible repressor binds back to its DNA-binding site, but since activator B is still present, autoactivation is achieved, resulting in a stable state. (C) and (D) depict the switch switched to state two and the function of its other positive feedback loop. <br />
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<h3>Modeling</h3><br />
<p>Before proceeding with experimental verification, we performed a <a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch">thorough modeling analysis</a> of the designed switch. We incorporated parameters obtained from the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators">repression and activation experimental results</a> into our simulations. We also implemented a new modular, hybrid modeling algorithm that introduced stochasticity into an otherwise deterministic approach and enabled us to explicitly model competitive transcription factor binding and a limited number of binding site repeats. Modeling is described in details in the <a href="https://2012.igem.org/Team:Slovenia/Modeling">modeling section</a>. Both deterministic and stochastic simulations demonstrate that the positive feedback loop switch is <b>significantly more stable than the mutual repressor switch. </b> Most importantly, <b>it can exhibit bistability even without cooperativity</b> (Figure 5). This switch is also <b>more robust than the simple mutual repressor switch in regard to leaky expression. </b> </p><br />
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<b>Figure 5. Robustness of the positive feedback loop switch. </b> The positive feedback loop switch exhibited bistability even in the absence of cooperativity and for the minimal promoter's leaky transcription rate of 10% or constitutive promoter leaky transcription rate of 5%. In comparison, the same leaky transcription rate (with equal values of all other parameters) of 5% caused the mutual repressor switch (without the positive feedback loops) to exhibit no bistability unless high cooperativity (2 or more) was introduced into the model. <br />
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<h3>Construction and experimental testing of the bistability of the switch </h3><br />
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<p>For experimental implementation of the positive feedback loop toggle switch we introduced the following components into cells (Figure 6): </p><br />
<ul style="padding-left:30px;"><br />
<li>a pair of <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB repressors</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a>), controlled by the opposite TAL (TALA controls the transcription of TALB and TALB controls the transcription of TALA), exactly as in the mutual repressor switch,</li><br />
<li>a pair of <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 activators</a> (TALA and TALB), each activating its own transcription (autoactivator) and transcription of the opposite TAL repressor ,</li><br />
<li>two of the constructs were tagged with fluorescent reporter proteins (BFP and mCitrine) via a t2A sequence, which ensured the equimolar production of the fluorescent reporter and TAL regulator,</li><br />
<li>Both TAL repressor and activator pairs (A and B), controlled by inducible repressors,</li><br />
<li>Constitutively expressed inducible repressor constructs,</li><br />
<li>Inducer molecules (pristinamycin and erythromycin) .</li><br />
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<p>We analyzed the performance of the switch by using two fluorescent proteins with a sufficientspectral distance (<b>BFP and mCitrine</b>), which enabled easy detection and quantification by confocal microscopy and flow cytometry. </p><br />
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<p>To analyze the bistability we first used flow cytometry, a technique which has a <b>unique ability to determine the number of cells expressing either one or both of the fluorescent reporter proteins. </b> Although cells were transfected with the complete switch device including both reporters, the analysis of cells demonstrated <b>clear bimodal distribution</b> - the majority of the transfected noninduced cells expressed only one of the two fluorescent proteins.The expression of one or the other reporter proteins is most likely a result of stochastic events or possibly a slight imbalance of the amount of transfected plasmids (Figure 7A).Importantly, a very low number of cells expressed both reporters. This bimodal distribution of fluorescence <b>clearly demonstrates the intrinsic bistability of our system</b> in comparison to the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch">mutual repressor switch</a> (classical toggle) topology, where a large fraction of cells expressed both <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch#gangnam">fluorescent reporters</a>. The addition of either one of the inducers switched the reporter production towards the corresponding fluorescent protein (Figures 7B and 7C). </p><br />
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<p><b>Figure 7. The bistable switch with a positive feedback loop exhibits a bimodal distribution of fluorescence. </b> (A) Non-induced HEK293 cells transfected with plasmids forming the switch (see Figure 6) transcribe either BFP or mCitrine as determined by flow cytometry. (B) HEK293T with the switch plasmids induced with erythromycin (2 mg/L) express mainly mCitrine and (C) cells induced with pristinamycin (2 mg/L) express BFP. Samples were analyzed five days after induction. </p><br />
<p><i>NOTE: A high fraction of cells in the lower left quadrant, typically between 50-70%,representsnontransfected cells.This is a typical fraction in mammalian cell transfection experiments and will be solved for the therapeutic use by the preparation of stable cell lines, whose selection however typically takes several months. </i></p><br />
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<p>Confocal microscopy confirmed <b>high expression of the expected and no expression of the opposite fluorescent reporter protein in both induced states</b> (Figure 8). This means the addition of an inducer shifts cells to a corresponding state which is <b>preserved even after the inducer has been removed</b> (Figure 9). The system remained in a stable state several days after the removal of the signal, which further confirms the epigenetic bistability of our positive feedback loop switch. </p><br />
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<b>Figure 8. Positive feedback loop switch exhibiting two different states at induction with pristinamycin (PI) and erythromycin (ER) inducer molecules. </b> HEK293T cells were cotransfected with the following plasmids : PCMV_mCherry transfection control (20 ng), [A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). Pristinamycin and erythromycin were added to final concentration of 2 µg/ml. Fluorescence was measured 3 days after induction. <br />
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<b>Figure 9. Positive feedback loop switch exhibiting stable states at removal of inducer molecules. </b><br/> HEK293T cells were cotransfected with the following plasmids: PCMV_mCherry (20 ng), [A]_PMIN_TALB:KRAB, <br/>[A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). Pristinamycin and erythromycin were added at final concentration of 2 µg/ml. Medium was replaced 3 days after induction and fluorescence was measured 3 days after removal of inducers. <br />
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<h2><b><font color="red">(NEW)</font></b> Switching between the two states</h2><br />
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<p>The <b>ultimate test</b> of the functionality of the bistable switch with a positive feedback loop is to <b>switch the system between the two states</b> by the addition of the second inducer. This is a longer experiment as it requires the system to settle first into the initial selected state by the addition of the first inducer and then switch into the second state after the addition of the second inducer. Nevertheless we managed to demonstrate this functionality of our switch during the time between the regional and world jamboree using a secretory luciferase assay and fluorescence microscopy.</p><br />
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<h3>Results</h3><br />
<p>After the induction of state I with pristinamycin we observed expression of reporter I, i.e. blue fluorescent protein (BFP) (Figure 10). Two days after the induction with pristinamycin, inducer was removed and replaced with erythromycin. We observed expression of the reporter mCitrine, an indicator of state II of the switch only one day after inducer exchange. We can still see some BFP emission in the next few days due to a relatively long half-life, but it is clear that mCitrine expression eventually prevails. </p><br />
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<b>Figure 10. Switching from state I to state II determined by confocal microscopy. </b> Expression of mCitrine was induced only after the addition of erythromycin.<br/> HEK293T cells were transfected with PCMV_mCherry (20 ng), [A]_PMIN_TALB:KRAB, [B]_PMIN_TALA:KRAB_t2A_mCitrine (10ng), [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALB:VP16, (2 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[ETR]_TALA:KRAB (20 ng), PCMV_[PIR]_TALA:VP16, CMV_[ETR]_TALB:VP16, (5 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (200 ng). Pristinamycin (2 µg/ml) was added to the cells to induce state I. After two days pristinamycin was removed and replaced with erythromycin to switch to state II. Each day images of cells were recorded with a confocal microscope. mCherry was used as a transfection control. <br />
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<p>Switching was also demonstrated using a cytosolic firefly luciferase (fLuc) and a secreted alkaline phosphatase ( SEAP) as state I and state II reporters. Secretory Renilla luciferase was used for normalization. Cells were induced with either erythromycin (2 mg/L) to trigger expression of fLuc or with pristinamycin (2 mg/L) to trigger expression of SEAP. Inducer I was removed after one day and replaced with inducer II. Expression of reporters was analyzed 3 and 5 days after inducer exchange. We observed reduced <b>expression of firefly luciferase and elevated expression of SEAP when pristinamycin was replaced by erythromycin</b> and <i>vice versa</i> when the cells were first stimulated with erythromycin and then with pristinamycin (Figure 11). </p><br />
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<b>Figure 11: Switching monitored by the activity of SEAP and firefly luciferase. HEK293T cells were co-transfected with plasmids of the switch and10x[B]_PCMV_fLuc and 10x[A]_CMV_SEAP reporter plasmids. Left, switch from pristinamycin to erythromycin state. Right, switch from erythromycin to pristinamycin induced state.</b><br />
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<p>Our system has considerable inertia as first the transcriptional factors must be degraded as well as the reporter proteins.The degradation could be accelerated by the addition of PEST degradation signals, which we already anticipated and prepared parts with TAL regulators that include pest signals. These regulators exhibit increased fold induction but have not yet been tested within the switch context. </p><br />
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<br /><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Cherry, J., L., Adler, F., R. (2000) How to make a Biological Switch. <i>J. Theor. Biol.</i> <b>203</b>, 117-133.<br />
<br/><br/><br />
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains.<i>Nat Commun.</i>3,968.<br /><br /><br />
Macía, J., Widder, S., Solé, R. (2009) Why are cellular switches Boolean? General conditions for multistable genetic circuits. <i>J. Theor. Biol.</i> <b>261</b>, 126-135. <br />
<br/><br/><br />
Widder, S., Macía, J., and Solé, R. (2009) Monomeric Bistability and the Role of Autoloops in Gene Regulation. <i>PLoS ONE</i> <b>4</b>, e5399.<br />
</p><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch
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2012-10-26T19:51:42Z
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<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<h1> Positive feedback loop switch</h1><br />
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<p>We designed an upgraded <b>bistable genetic toggle switch based on orthogonal TAL repressors and activators</b> which is composed of a pair of mutual repressors and a pair of activators,upgradingthe classical toggle switch with positive feedback loop. </p><br />
<p>Simulations of the positive feedback loop switch demonstrated <b>bistability even at low or no cooperativity</b>. </p><br />
<p>Stochastic and deterministic simulations indicate <b>higher robustness</b> in comparison to the mutual repressor switch. </p> <br />
<p>We experimentally tested the switch by monitoring production of two fluorescent protein reporters, confirming a <b>clear bimodal distribution of reporter fluorescence</b> and demonstrated adoption of <b>stable states</b> by induction with corresponding inducer molecules. </p><br />
<p>The switch <b>persisted in a stable state</b> after the removal of inducer molecules, which confirmed the epigenetic bistability of our system. </p><br />
<p><b><font color="red">(NEW)</font></b> Cells could <b>switch from one state to the other</b> by the addition of the second inducer, which was confirmed by two methods. </p> <br />
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<h2>Bistable genetic switch based on non-cooperative elements</h2><br />
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<p>Mathematical analysis of genetic switches from the literature indicates that cooperativity, which introduces a nonlinear response, is required for a functional bistable switch, consisting of two mutual repressors (Cherry et al. 2000). Macia et al. (2009) and Widder et al. (2009) proposed that <b>bistability could be introduced by non-cooperative elements, when nonlinearity is introduced</b> for exampleif protein A is able to repress the transcription of protein B and at the same time activate its own transcription and <i>vice versa</i> (Figure 1). </p> <br />
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<td class="invisible" style="text-align:justify; width:70%;"> <b>Figure 1. Macia et al. (2009) and Widder et al. (2009) proposed a mathematical solution for creation of a bistable switch that would not require the cooperative binding of transcriptional regulators. </b> According to the proposed model, transcription factor A should activate its own transcription and repress the transcription of gene B and <i>vice versa.</i> <br />
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<p>We, as molecular biologists, werenot aware of a transcriptional effector or effector pair that could act simultaneously as a repressor and activator, therefore we were not surprised that, to our knowledge, this type of bistable switch has not yet been experimentally implemented. </p> <br />
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<p>The crucial moment in our project was when we realized that the ability to design <b>TAL repressors and activators directed against the same binding site could offer a solution to this problem and provide a unique opportunity to construct orthogonal bistable switches based on noncooperative elements (Figure 2). </b> </p><br />
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<td class="invisible" style="text-align:justify; width:100%;"> <b>Figure 2. A repressor (left) and an activator (right) competing for the same binding site. </b> <br />
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<h2>Results</h2><br />
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<h3>Design</h3><br />
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<p>We designed an <b>upgraded mutual repressor switch, </b> introducing <b>two additional positive feedback loops</b> (Figure 3), consisting of two TAL activators targeted against the same binding sites as apair of mutual TAL repressors. In other words, <b>rather than having the same protein function as an activator and repressor, we used an activator and repressor pair, competing for the same operator. </b> The same binding sequence for the activator and repressor introduced nonlinearity required for the bistability based oncompetition for the binding site. For the purpose of our project, <b>we designed a bistable switch with a positive feedback loop, capable of switching between the two states through regulation by inducer molecules</b> (Figure 4). </p><br />
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<b>Figure 3. Scheme of the genetic circuit for the bistable toggle switch composed of a pair of mutual repressors and a pair of autoactivators under the control of two operators. </b><br />
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<b>Figure 4. Designed states of the bistable toggle switch with a positive feedback loop. </b> (A) Addition of inducer 1 triggers dissociation of its cognate repressor from its DNA-binding site. As a consequence, repressor A and activator B are transcribed. Activator B further activates transcription of repressor A and itself, which forms the positive feedback loop of the switch state one. Meanwhile repressor A inhibits the other state of the switch. (B) When the inducer is removed, the inducible repressor binds back to its DNA-binding site, but since activator B is still present, autoactivation is achieved, resulting in a stable state. (C) and (D) depict the switch switched to state two and the function of its other positive feedback loop. <br />
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<h3>Modeling</h3><br />
<p>Before proceeding with experimental verification, we performed a <a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch">thorough modeling analysis</a> of the designed switch. We incorporated parameters obtained from the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators">repression and activation experimental results</a> into our simulations. We also implemented a new modular, hybrid modeling algorithm that introduced stochasticity into an otherwise deterministic approach and enabled us to explicitly model competitive transcription factor binding and a limited number of binding site repeats. Modeling is described in details in the <a href="https://2012.igem.org/Team:Slovenia/Modeling">modeling section</a>. Both deterministic and stochastic simulations demonstrate that the positive feedback loop switch is <b>significantly more stable than the mutual repressor switch. </b> Most importantly, <b>it can exhibit bistability even without cooperativity</b> (Figure 5). This switch is also <b>more robust than the simple mutual repressor switch in regard to leaky expression. </b> </p><br />
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<b>Figure 5. Robustness of the positive feedback loop switch. </b> The positive feedback loop switch exhibited bistability even in the absence of cooperativity and for the minimal promoter's leaky transcription rate of 10% or constitutive promoter leaky transcription rate of 5%. In comparison, the same leaky transcription rate (with equal values of all other parameters) of 5% caused the mutual repressor switch (without the positive feedback loops) to exhibit no bistability unless high cooperativity (2 or more) was introduced into the model. <br />
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<h3>Construction and experimental testing of the bistability of the switch </h3><br />
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<p>For experimental implementation of the positive feedback loop toggle switch we introduced the following components into cells (Figure 6): </p><br />
<ul style="padding-left:30px;"><br />
<li>a pair of <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB repressors</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a>), controlled by the opposite TAL (TALA controls the transcription of TALB and TALB controls the transcription of TALA), exactly as in the mutual repressor switch,</li><br />
<li>a pair of <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 activators</a> (TALA and TALB), each activating its own transcription (autoactivator) and transcription of the opposite TAL repressor ,</li><br />
<li>two of the constructs were tagged with fluorescent reporter proteins (BFP and mCitrine) via a t2A sequence, which ensured the equimolar production of the fluorescent reporter and TAL regulator,</li><br />
<li>Both TAL repressor and activator pairs (A and B), controlled by inducible repressors,</li><br />
<li>Constitutively expressed inducible repressor constructs,</li><br />
<li>Inducer molecules (pristinamycin and erythromycin) .</li><br />
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<p>We analyzed the performance of the switch by using two fluorescent proteins with a sufficientspectral distance (<b>BFP and mCitrine</b>), which enabled easy detection and quantification by confocal microscopy and flow cytometry. </p><br />
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<p>To analyze the bistability we first used flow cytometry, a technique which has a <b>unique ability to determine the number of cells expressing either one or both of the fluorescent reporter proteins. </b> Although cells were transfected with the complete switch device including both reporters, the analysis of cells demonstrated <b>clear bimodal distribution</b> - the majority of the transfected noninduced cells expressed only one of the two fluorescent proteins.The expression of one or the other reporter proteins is most likely a result of stochastic events or possibly a slight imbalance of the amount of transfected plasmids (Figure 7A).Importantly, a very low number of cells expressed both reporters. This bimodal distribution of fluorescence <b>clearly demonstrates the intrinsic bistability of our system</b> in comparison to the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch">mutual repressor switch</a> (classical toggle) topology, where a large fraction of cells expressed both <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch#gangnam">fluorescent reporters</a>. The addition of either one of the inducers switched the reporter production towards the corresponding fluorescent protein (Figures 7B and 7C). </p><br />
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<p><b>Figure 7. The bistable switch with a positive feedback loop exhibits a bimodal distribution of fluorescence. </b> (A) Non-induced HEK293 cells transfected with plasmids forming the switch (see Figure 6) transcribe either BFP or mCitrine as determined by flow cytometry. (B) HEK293T with the switch plasmids induced with erythromycin (2 mg/L) express mainly mCitrine and (C) cells induced with pristinamycin (2 mg/L) express BFP. Samples were analyzed five days after induction. </p><br />
<p><i>NOTE: A high fraction of cells in the lower left quadrant, typically between 50-70%,representsnontransfected cells.This is a typical fraction in mammalian cell transfection experiments and will be solved for the therapeutic use by the preparation of stable cell lines, whose selection however typically takes several months. </i></p><br />
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<p>Confocal microscopy confirmed <b>high expression of the expected and no expression of the opposite fluorescent reporter protein in both induced states</b> (Figure 8). This means the addition of an inducer shifts cells to a corresponding state which is <b>preserved even after the inducer has been removed</b> (Figure 9). The system remained in a stable state several days after the removal of the signal, which further confirms the epigenetic bistability of our positive feedback loop switch. </p><br />
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<b>Figure 8. Positive feedback loop switch exhibiting two different states at induction with pristinamycin (PI) and erythromycin (ER) inducer molecules. </b> HEK293T cells were cotransfected with the following plasmids : PCMV_mCherry transfection control (20 ng), [A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). Pristinamycin and erythromycin were added to final concentration of 2 µg/ml. Fluorescence was measured 3 days after induction. <br />
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<b>Figure 9. Positive feedback loop switch exhibiting stable states at removal of inducer molecules. </b><br/> HEK293T cells were cotransfected with the following plasmids: PCMV_mCherry (20 ng), [A]_PMIN_TALB:KRAB, <br/>[A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). Pristinamycin and erythromycin were added at final concentration of 2 µg/ml. Medium was replaced 3 days after induction and fluorescence was measured 3 days after removal of inducers. <br />
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<h2>Switching between the two states NEW</h2><br />
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<p>The <b>ultimate test</b> of the functionality of the bistable switch with a positive feedback loop is to <b>switch the system between the two states</b> by the addition of the second inducer. This is a longer experiment as it requires the system to settle first into the initial selected state by the addition of the first inducer and then switch into the second state after the addition of the second inducer. Nevertheless we managed to demonstrate this functionality of our switch during the time between the regional and world jamboree using a secretory luciferase assay and fluorescence microscopy.</p><br />
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<h3>Results</h3><br />
<p>After the induction of state I with pristinamycin we observed expression of reporter I, i.e. blue fluorescent protein (BFP) (Figure 10). Two days after the induction with pristinamycin, inducer was removed and replaced with erythromycin. We observed expression of the reporter mCitrine, an indicator of state II of the switch only one day after inducer exchange. We can still see some BFP emission in the next few days due to a relatively long half-life, but it is clear that mCitrine expression eventually prevails. </p><br />
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<b>Figure 10. Switching from state I to state II determined by confocal microscopy. </b> Expression of mCitrine was induced only after the addition of erythromycin.<br/> HEK293T cells were transfected with PCMV_mCherry (20 ng), [A]_PMIN_TALB:KRAB, [B]_PMIN_TALA:KRAB_t2A_mCitrine (10ng), [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALB:VP16, (2 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[ETR]_TALA:KRAB (20 ng), PCMV_[PIR]_TALA:VP16, CMV_[ETR]_TALB:VP16, (5 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (200 ng). Pristinamycin (2 µg/ml) was added to the cells to induce state I. After two days pristinamycin was removed and replaced with erythromycin to switch to state II. Each day images of cells were recorded with a confocal microscope. mCherry was used as a transfection control. <br />
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<p>Switching was also demonstrated using a cytosolic firefly luciferase (fLuc) and a secreted alkaline phosphatase ( SEAP) as state I and state II reporters. Secretory Renilla luciferase was used for normalization. Cells were induced with either erythromycin (2 mg/L) to trigger expression of fLuc or with pristinamycin (2 mg/L) to trigger expression of SEAP. Inducer I was removed after one day and replaced with inducer II. Expression of reporters was analyzed 3 and 5 days after inducer exchange. We observed reduced <b>expression of firefly luciferase and elevated expression of SEAP when pristinamycin was replaced by erythromycin</b> and <i>vice versa</i> when the cells were first stimulated with erythromycin and then with pristinamycin (Figure 11). </p><br />
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<b>Figure 11: Switching monitored by the activity of SEAP and firefly luciferase. HEK293T cells were co-transfected with plasmids of the switch and10x[B]_PCMV_fLuc and 10x[A]_CMV_SEAP reporter plasmids. Left, switch from pristinamycin to erythromycin state. Right, switch from erythromycin to pristinamycin induced state.</b><br />
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<p>Our system has considerable inertia as first the transcriptional factors must be degraded as well as the reporter proteins.The degradation could be accelerated by the addition of PEST degradation signals, which we already anticipated and prepared parts with TAL regulators that include pest signals. These regulators exhibit increased fold induction but have not yet been tested within the switch context. </p><br />
<br />
<br /><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Cherry, J., L., Adler, F., R. (2000) How to make a Biological Switch. <i>J. Theor. Biol.</i> <b>203</b>, 117-133.<br />
<br/><br/><br />
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains.<i>Nat Commun.</i>3,968.<br /><br /><br />
Macía, J., Widder, S., Solé, R. (2009) Why are cellular switches Boolean? General conditions for multistable genetic circuits. <i>J. Theor. Biol.</i> <b>261</b>, 126-135. <br />
<br/><br/><br />
Widder, S., Macía, J., and Solé, R. (2009) Monomeric Bistability and the Role of Autoloops in Gene Regulation. <i>PLoS ONE</i> <b>4</b>, e5399.<br />
</p><br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'>Controls >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T19:37:46Z
<p>Dusanv: </p>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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</ul><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href="https://2012.igem.org/Team:Slovenia/Parts#TALs">TAL regulators</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005">TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href="https://2012.igem.org/Team:Slovenia/Parts#reporters">reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href="http://partsregistry.org/Part:BBa_K323214">NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href="http://partsregistry.org/Part:BBa_K782007">NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<h3>Designed repressors</h3><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009">KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011">NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<h3><b><font color="red">(NEW)</font></b> TAL regulators are functional and nontoxic for multicellular animal</h3><br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrine under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter. It is exciting to see fluorescent tadpoles that were transfected with functional TAL activator and a reporter, which demonstrates that TAL activator is not deleterious for the development of the animal.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed 1 and 3 days after introduction of plasmids under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results obtained in collaboration with <a href="https://2012.igem.org/Team:Evry">Evry team</a> (thank you froggies) thus demonstrate that TAL-based logic can be used also in the whole animal, which could be used as an excellent model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
<br/><br/><br />
Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
<br/><br/><br />
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
<br/><br/><br />
Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
<br/><br/><br />
Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
<br/><br/><br />
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
<br/><br/><br />
Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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</p><br />
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<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
</b><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T19:22:58Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href="https://2012.igem.org/Team:Slovenia/Parts#TALs">TAL regulators</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005">TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href="https://2012.igem.org/Team:Slovenia/Parts#reporters">reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href="http://partsregistry.org/Part:BBa_K323214">NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href="http://partsregistry.org/Part:BBa_K782007">NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<h3>Designed repressors</h3><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009">KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB</a> fusions. </p><br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011">NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<h3><b><font color="red">NEW</font></b> TAL regulators are functional and nontoxic for multicellular animal</h3><br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrine under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter. It is exciting to see fluorescent tadpoles that were transfected with functional TAL activator and a reporter, which demonstrates that TAL activator is not deleterious for the development of the animal.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed 1 and 3 days after introduction of plasmids under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results obtained in collaboration with <a href="https://2012.igem.org/Team:Evry">Evry team</a> (thank you froggies) thus demonstrate that TAL-based logic can be used also in the whole animal, which could be used as an excellent model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T19:22:35Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
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</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br/><br />
<br />
<h1> TAL-based transcriptional regulators</h1><br />
<br />
<table class="summary"><br />
<tr class="summary"><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href="https://2012.igem.org/Team:Slovenia/Parts#TALs">TAL regulators</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005">TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href="https://2012.igem.org/Team:Slovenia/Parts#reporters">reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href="http://partsregistry.org/Part:BBa_K323214">NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href="http://partsregistry.org/Part:BBa_K782007">NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009">KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011">NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<h3><b><font color="red">NEW</font></b>TAL regulators are functional and nontoxic for multicellular animal</h3><br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrine under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter. It is exciting to see fluorescent tadpoles that were transfected with functional TAL activator and a reporter, which demonstrates that TAL activator is not deleterious for the development of the animal.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed 1 and 3 days after introduction of plasmids under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results obtained in collaboration with <a href="https://2012.igem.org/Team:Evry">Evry team</a> (thank you froggies) thus demonstrate that TAL-based logic can be used also in the whole animal, which could be used as an excellent model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T19:11:33Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
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<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
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<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href="https://2012.igem.org/Team:Slovenia/Parts#TALs">TAL regulators</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005">TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href="https://2012.igem.org/Team:Slovenia/Parts#reporters">reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href="http://partsregistry.org/Part:BBa_K323214">NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href="http://partsregistry.org/Part:BBa_K782007">NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009">KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011">NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrine under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter. It is exciting to see fluorescent tadpoles that were transfected with functional TAL activator and a reporter, which demonstrates that TAL activator is not deleterious for the development of the animal.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed 1 and 3 days after introduction of plasmids under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results obtained in collaboration with <a href="https://2012.igem.org/Team:Evry">Evry team</a> (thank you froggies) thus demonstrate that TAL-based logic can be used also in the whole animal, which could be used as an excellent model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T19:10:50Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href="https://2012.igem.org/Team:Slovenia/Parts#TALs">TAL regulators</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005">TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href="https://2012.igem.org/Team:Slovenia/Parts#reporters">reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href="http://partsregistry.org/Part:BBa_K323214">NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href="http://partsregistry.org/Part:BBa_K782007">NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<h3>Designed repressors</h3><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009">KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011">NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrin under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter. It is exciting to see fluorescent tadpoles that were transfected with functional TAL activator and a reporter, which demonstrates that TAL activator is not deleterious for the development of the animal.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed 1 and 3 days after introduction of plasmids under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results obtained in collaboration with <a href="https://2012.igem.org/Team:Evry">Evry team</a> (thank you froggies) thus demonstrate that TAL-based logic can be used also in the whole animal, which could be used as an excellent model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
<br/><br/><br />
Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
<br/><br/><br />
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
<br/><br/><br />
Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
<br/><br/><br />
Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/File:Svn12_evryfrogs.png
File:Svn12 evryfrogs.png
2012-10-26T18:43:12Z
<p>Dusanv: uploaded a new version of &quot;File:Svn12 evryfrogs.png&quot;</p>
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Dusanv
http://2012.igem.org/Team:Slovenia/Notebook
Team:Slovenia/Notebook
2012-10-26T17:40:16Z
<p>Dusanv: </p>
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<br/><br />
<h1><a name="naslov"></a>Experimental methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#cloning">Cloning</a><br/></li><br />
<li><a href="#cellcultures">Cell cultures</a></li><br />
<li><a href="#inductionsystems">Induction systems</a></li><br />
<li><a href="#effectors">Effectors</a></li><br />
<li><a href="#microscopy">Microscopy</a></li><br />
<li><a href="#flowcytometry">Flow cytometry</a></li><br />
<li><a href="#microencapsulation">Microencapsulation</a></li><br />
<li><a href="#proteindetection">Protein detection</a></li><br />
</ul><br />
</p><br />
<br />
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<td class="invisible"><b>Figure 1.</b> Schematic presentation of methods used for cloning and culturing eukaryotic cells.<br />
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<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/8/84/SVN12_7_notebook_switch.png"/></td><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/e/e7/SVN12_7_notebook_safety_mech.png"/></td><br />
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<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/9/91/SVN12_7_notebook_encapsulation.png"/></td><br />
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<td class="invisible"><b>Figure 2.</b> Schematic presentation of methods used for characterizing the switch, safety mechanisms, microencapsulation and effectors.</td><br />
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</tbody><br />
</table><br />
<br />
<h2><a name="cloning"> Cloning </a></h2><br />
<h3>Plasmid DNA isolation</h3><br />
<p><b>MINI PREPs for analysis and sequencing</b></p><br />
<ol><br />
<li>A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). </li><br />
<li>Bacteria were grown over night at 37 °C with agitation. </li><br />
<li>Plasmid DNA was isolated from 6-10 mL of over-night culture with GeneJET plasmid miniprep kit according to the manufacturer's protocol. </li><br />
<li>Amounts ranging from 6-10 µg of plasmid DNA were obtained. </li><br />
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Fragment DNA isolation from agarose gel</h3><br />
<p><b>AGAROSE ELECTROPHORESIS</b></p><br />
<ol><br />
<li>A mixture of different sized DNA fragments was separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li><br />
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li><br />
</ol><br />
</br><br />
<p><b>FRAGMENT ISOLATION from agarose gel </b></p><br />
<ol><br />
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li><br />
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li><br />
<li>Purity and amount of DNA was determined using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Restriction digest</h3><br />
<ol><br />
<li>To digest the desired DNA restriction reactions were prepared as follows: </li><br />
<ul style="margin-left:15px;"> <b>for analysis of cloned DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>0.5 µL restriction enzyme</li><br />
<br />
<li>Bring volume to 20 µL with nuclease-free water.</li><br />
</ul><br />
<i>or</i><br />
<ul style="margin-left:15px;"> <b>for isolation of specific DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>up to 2 µL restriction enzyme </li><br />
<br />
<li>Bring volume to 50 µL with nuclease-free water.</li><br />
</ul><br />
</li><br />
<li>The sample was incubated at optimal temperature for the restriction enzymes.</li><br />
<li>Analysis of fragmented DNA was done by gel electrophoreses. </li><br />
<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<h3>PCR reaction</h3><br />
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p><br />
<ol><br />
<br />
<li>The master mix for reactions with Phusion DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (1-10 ng)</li><br />
<br />
<li>both primers (0,4 pmol/µl )</li><br />
<br />
<li>1x Phusion HF buffer</li><br />
<br />
<li>0,2 µM dNTPs</li><br />
<br />
<li>Phusion polymerase (0,02 U/ µl) and </li><br />
<br />
<li>MQ up to final volume of 25 µl</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with AccuPrime DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (10 ng),</li><br />
<br />
<li>both primers (0,4 pmol/µl ),</li><br />
<br />
<li>1xRnx mix,</li><br />
<br />
<li>enzyme (0,05 U/ µl) and</li><br />
<br />
<li>MQ up to final volume of 50 µl.</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with Taq DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>both primers (0,4 pmol/µl),</li><br />
<br />
<li>1x Taq PCR buffer II,</li><br />
<br />
<li>0,2 µM dNTPs,</li><br />
<br />
<li>5mM MgSO4,</li><br />
<br />
<li>enzyme (0,125 U/ µl) and</li><br />
<br />
<li>MQ up to total volume of 20 µl.</li><br />
<br />
<li>Then the bacterial colony was added to the reaction mix.</li> <br />
</ul><br />
</li><br />
<br />
<li>All temperature profiles were optimized according to manufacturer’s protocol,the melting temperature of primers, and the length of the desired PCR products. Reactions were performed in the Applied Biosystems Veriti 96 well thermal cycler. </li><br />
</ol><br />
</br><br />
<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p><br />
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p><br />
<h3>Gibson assembly </h3><br />
<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p><br />
<ol><br />
<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li><br />
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Ligation</h3><br />
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p><br />
<ol><br />
<li>Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA). </li><br />
<li>A ligation mixture was prepared: </li><br />
<br/><br />
1X ligase buffer (10X)<br />
<br/><br />
1 µL T4 ligase (3 U/µL)<br />
<br/><br />
Bring volume to 10 or 20 µL with nuclease-free water.<br />
</li></br><br />
<i>or</i><br />
<br />
<li>Blunt-end ligation reactions were incubated at 17 °C for 4 to 18 hours. </li><br />
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li><br />
</ol><br />
</br><br />
<br />
<h3> Culturing bacteria</h3><br />
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p><br />
<p><b> Growth media for bacteria</b></p><br />
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p><br />
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p><br />
</br><br />
<br />
<h3>Transformation of bacteria</h3><br />
<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p><br />
<ol><br />
<li>100 µL of competent cells were thawed on ice. </li><br />
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li><br />
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li><br />
<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li><br />
<li>Cooled for 3 minutes on ice. </li><br />
<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li><br />
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li><br />
</ol><br />
</br><br />
<br />
<h3>Glycerol stock for long term storage of bacteria</h3><br />
<ol><br />
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li><br />
<li>Mixed and incubated at room temperature for 30 minutes. </li><br />
<li>Afterwards the glycerol stock was stored at -80 °C. </li><br />
</ol><br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="cellcultures"> Cell cultures </a></h2><br />
<h3>Eucaryotic cell lines and cultivation</h3><br />
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p><br />
</br><br />
<br />
<h3>Subculturing monolayer cell cultures</h3><br />
<ol><br />
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li><br />
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li><br />
<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li><br />
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li><br />
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li><br />
<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li><br />
<li>Remove the trypsin-containing medium from the centrifuge tube. </li><br />
<li>Resuspend the cell pellet in fresh medium. </li><br />
<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li><br />
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li><br />
</ol><br />
</br><br />
<h3>Cell plating</h3><br />
<br />
<ol><br />
<li>Count cells. </li><br />
<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li><br />
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li><br />
</ol><br />
<br/><br />
<p><b>Media and buffers</b><br />
<br/><br />
<br><b>DMEM</b> supplemented with: 1 % L-Glutamine (GlutaMax), 10 % FBS, Optionally: 1% Pen/Strep.<br />
<br><b>RPMI</b> supplemented with: 1 % L-Glutamine (GlutaMax), 20 % FBS.</p><br />
<br />
<br/><br />
<h3>Transfection</h3><br />
TABLE: Transfection mixtures for different culture format<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Culture format</th><br />
<th>jetPEI reagent per µg of DNA (µL)</th><br />
<th>Typical amount of DNA (ng)</th><br />
<th>Volume of 150 mM NaCl solution for DNA and jetPEI (µL)</th><br />
<th>Total transfection mixture volume (µL)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">96-well</td><br />
<td class="normal">2</td><br />
<td class="normal">200</td><br />
<td class="normal">10</td><br />
<td class="normal">20</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">24-well and 8-well microscope chamber</td><br />
<td class="normal">2</td><br />
<td class="normal">500</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal">12-well</td><br />
<td class="normal">2</td><br />
<td class="normal">1000</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">6-well</td><br />
<td class="normal">2</td><br />
<td class="normal">2000</td><br />
<td class="normal">100</td><br />
<td class="normal">200</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">10 cm</td><br />
<td class="normal">2</td><br />
<td class="normal">15000</td><br />
<td class="normal">250</td><br />
<td class="normal">500</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<ol><br />
<li>Dilute plasmid DNA to desired concentration in 150 mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Dilute jetPEI (PolyPlus) in 150mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Add the jetPEI solution to the DNA solution. </li><br />
<li>Vortex the solution immediately and spin down briefly. </li><br />
<li>Incubate for 15 to 30 minutes at room temperature. </li><br />
<li>Add the jetPEI/DNA mix to the cells in and gently swirl the plate. </li><br />
<li>Return the plate to a cell culture incubator. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="inductionsystems"> Induction systems </a></h2><br />
<br />
<h3>Induction systems</h3><br />
<br />
<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p><br />
<br />
<h3>Tetracycline, erythromycin and pristinamycin systems</h3><br />
<br />
<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Svn12_parts_inducibilni.png"></img><br />
<center><p><b> Figure 1: Induced expression of TAL</b>.</p></center><br />
<br />
<p>TABLE</p><br />
<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> System </th><br />
<br />
<th> Tetracycline </th><br />
<br />
<th> Erythromycin</th><br />
<br />
<th> Pristinamycin </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> Regulating protein </td><br />
<td class="normal"> TetR:KRAB </td><br />
<td class="normal"> E:KRAB </td><br />
<td class="normal"> PIP:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> TRE </td><br />
<td class="normal"> ETR </td><br />
<td class="normal"> PIR </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> Inducer </td><br />
<td class="normal"> Tetracycline or doxycycline </td><br />
<td class="normal"> Erythromycin </td><br />
<td class="normal"> Pristinamycin </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> pCMV_TRE_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_ETR_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> pCMV_TRE_TAL-B:VP16</td><br />
<td class="normal"> pCMV_ETR_TAL-B:VP16</td><br />
<td class="normal"> pCMV_PIR_TAL-A:VP16</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-B:KRAB </td><br />
<td class="normal"> pSV40_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-A:VP16 </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pSV40_ETR_TAL-B:KRAB </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<h3>Rapamycin system</h3><br />
<br />
<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p><br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> Regulating vector </th><br />
<br />
<th> HetAct </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> ZFHD </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> ZFHD_pMIN_TAL-A:KRAB </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> ZFHD_pMIN_TAL-B:VP16</td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
</br><br />
<h3>Induction of cells</h3><br />
<ol><br />
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li><br />
<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li><br />
</ol><br />
</br><br />
<br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Inductor</th><br />
<th>Stock solution (solvent)</th><br />
<th>Dilution (solvent)</th><br />
<th>Concentration in cell medium (% solvent)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">Rapalogue (AP21967)</td><br />
<td class="normal">1 mM (100% ethanol)</td><br />
<td class="normal">10 µM (1% ethanol)</td><br />
<td class="normal">1 µM (0,1% ethanol)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Doxycyclin</td><br />
<td class="normal">1 g/L (MQ)</td><br />
<td class="normal">10 mg/L (MQ)</td><br />
<td class="normal">1 mg/L (MQ)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Pristinamycin</td><br />
<td class="normal">50 g/L (100% DMSO)</td><br />
<td class="normal">20 mg/L (1% DMSO)</td><br />
<td class="normal">2 mg/L (0,1% DMSO)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Erythromycin</td><br />
<td class="normal">50 g/L (100% ethanol)</td><br />
<td class="normal">20 mg/L (1% ethanol)</td><br />
<td class="normal">2 mg/L (0,1% ethanol)</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<!-- ----------------------------------------------------------------- --><br />
</br><br/><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="effectors"> Effectors </a></h2><br />
<h3>Biological assay-anakinra </h3><br />
<ol><br />
<li>HEK293T cells, seeded in 6-well plate, were transfected with anakinra downstream of constitutive promoter. </li><br />
<li>Transfected cells were incubated for 48 h. </li><br />
<li>To detect anakinra's effect on NF-κB signalling pathway, other HEK293T cells were transfected with plasmid coding for Renilla luciferase and plasmid reporter with NF-κB-inducible firefly luciferase expression. HEK293T cells express IL-1R, so additional transfection with a receptor gene was notneeded. </li><br />
<li>After 24 h, the medium was removed from cells transfected with reporter plasmids and 90 μL of anakinra-producing cells' supernatant was added to these wells. </li><br />
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li><br />
</ol><br />
</br><br />
<br />
<h3> Biological assay-IFN-alpha </h3><br />
<ol><br />
<li>HEK293T cells transfected with eithera plasmid encoding IFN-alpha under the control of a constitutive promoter or an empty vector, and HEK293T cells transfected with the reporter vector were co-cultivated . </li><br />
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li><br />
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li><br />
<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li><br />
</ol><br />
</br><br />
<br />
<h3>ELISA for IFN-alpha</h3><br />
<ol><br />
<li>HEK293T cells where plated on 6 well plates and transfected with a plasmid coding for human IFN-alpha under the control of a constitutive promoter or a control plasmid (pcDNA3). </li><br />
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-fluorescence(The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in black 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega ) per well. </li><br />
<li>Fluorescence was measured using an automated plate reader. </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-luminescence (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two to three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li><br />
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Plate reader-absorbance (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li><br />
<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li><br />
<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C the absorbance was measured at 630 nm. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="microscopy"> Microscopy </a></h2><br />
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p><br />
</br><br />
<br />
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3><br />
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 200 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li><br />
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 nm and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell growth (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng GFP (pPCMV-GFP) (for transfection control). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a cell cultures were imaged. </li><br />
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite GFP reporter protein. Fluorescence emission was detected at 520-560 nm for GFP. Bright field images were used to visualize the number of cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell count (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates and transfected with different amounts of mGMK:TK30 (pPCMV_mGMK:TK30) as indicated in Figure legend. </li><br />
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the Figure legend. </li><br />
<li>After incubation the cells were resuspended by pipetting. </li><br />
<li>Cells suspensions were then mixed with trypan blue. </li><br />
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-detection of reporter proteins (The Switch) </h3><br />
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p><br />
<ol><br />
<li>HEK293T cells were seeded in an 8-well microscope chamber or 12-well plate and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>Images of cells expressing reporters were taken two days after transfection and then each day for 5 days. </li><br />
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-alginate degradation (Microencapsulation) </h3><br />
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation upon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p><br />
<ol><br />
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into well. </li><br />
<li>After the dye was evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added. </li><br />
<li>The microscope was set to capture images every 20 seconds. </li><br />
<li>Screenshots were collected for at least 15 minutes. </li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Microscopy-secreted alginate lyase enzymatic activity (Microencapsulation)</h3><br />
<ol><br />
<li>HEK293T cells were seeded 1×10<sup>6</sup> on 10 cm cell culture dish and grown in DMEM medium supplemented with 10 % FBS.</li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection).</li><br />
<li>Protein production lasted for 72 hours.</li><br />
<li>Cell supernatants were collected and concentrated 50-times using Sartorius Vivaspin 6 concentrators.</li><br />
<li>Alginate beads were produced with Büchi BIOTECH Encapsulator (see Microencapsulation: Encapsulation procedure 1.-6.).</li><br />
<li>Beads were incubated with concentrated supernatants for 72 hours in an 8-well microscope chamber.</li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into wells.</li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC detection. Fluorescence emission was detected at 520-560 nm.</li><br />
<li>Beads' diameters were assessed using Leica LAS Image Analysis software.</li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3><br />
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both live and dead cells, while 7-AAD stains dead cells only.</p><br />
<ol><br />
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS. </li><br />
<li>200 µL of the microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber. </li><br />
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added to one well. </li><br />
<li>Encapsulated cells were protected from direct light and stained for 30 minutes at 37 °C. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 543-nm line of HeNe laser was used to excite 7-AAD. </li><br />
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="flowcytometry"> Flow cytometry </a></h2><br />
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p><br />
</br><br />
<br />
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labeled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with phosphatidylserine exposed on their surface.</p><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates. </li><br />
<li>Cells were transfected with pCMV-mGMK_TK30 and treated with ganciclovir. Concentrations of ganciclovir and plasmids are indicated in Figure legends. </li><br />
<li>After incubation the cells were washed with PBS buffer and resuspended by pipetting. </li><br />
<li>Cells were pelleted with centrifugation at 1200 rpm. </li><br />
<li>The cell pellet was washed in 1x Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). </li><br />
<li>The pellet was then resuspended in 1x Annexin Binding Buffer and PE-Annexin V (5 µl per 100 μl cell suspension) was added. </li><br />
<li>Samples were incubated for 20 minutes in the dark at room temperature and then immediately analyzed with a flow cytometer. </li><br />
<li>Along with site and forward scatter, the signal in the FL2 channel (540-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p><br />
<ol><br />
<li>HEK293T cells seeded in 12-well plates were transfected with plasmids expressing MICA (pPCMV-MICA_pcDNA3).</li><br />
<li>Two days after transfection with CMV-MICA_pcDNA3, cells were resuspended in PBS at final concentration 1x10<sup>6</sup> cells/mL, stained with CFSE (0,6μM) and incubated for 10 minutes at 37°C. Staining was quenched by the addition of 5 volumes of ice-cold culture media (RPMI+ 20% FBS) to the cells. After 5 minutes incubation on ice, cells were pelleted by centrifugation and then washed by resuspending the pellet in fresh media (RPMI+ 20% FBS) a further two times for a total of three washes. CFSE was used to discriminate between HEK293T and NK-92 cells or between NK target cells K562, which were used as a positive control, and NK-92 cells.</li><br />
<li>HEK293T cells or K562 cells were mixed with NK-92 cells in different ratios (1:1, 1:5, 1:10) and incubated for 4 hours at 37 °C in culture medium consisting of RPMI, 20% FBS and hIL-2 (100 U/ml).</li><br />
<li>After incubation of HEK293T or K562 cells with NK-92 cells, cells were treated with propidium iodide.</li><br />
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded.</li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3><br />
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p><br />
<ol><br />
<li>HEK293T cells were seeded in a 12 or 24-well plate and transfected with plasmids encoding the switch. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. </li><br />
<li>Medium with inducer or without the inducer was changed after two days of cultivation. </li><br />
<li>Cells were collected at different time points (2 days after induction and then 3 days after the second media change). </li><br />
<li>Cells were washed and resuspended in PBS buffer. </li><br />
<li>A 405 nm diode laser was used to excite tagBFP and a 488-nm diode laser was used for mCitirne. </li><br />
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="microencapsulation"> Microencapsulation </a></h2><br />
<h3>Cell preparation for encapsulation</h3><br />
<ol><br />
<li>HEK 293T cells were seeded 5x10⁵ per 10 cm cell culture dish (3 per encapsulation) and grown in DMEM medium supplemented with 10 % FBS. </li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection). </li><br />
<li>The medium was removed the next day.. Transfected cells were detached using 3 mL of trypsin solution and centrifuged after the addition of fresh medium to inactivate the trypsin. </li><br />
<li>Supernatant was removed and cells were resuspended in 15 mL DMEM with 10% FBS. </li><br />
<li>Cells were counted using Countess automated cell counter (Invitrogen). </li><br />
<li>HEK 293T cells were again centrifuged and supernatant was removed. </li><br />
<li>Cells were resuspended in 2 mL of pre-warmed MOPS buffer. </li><br />
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li><br />
</ol><br />
</br><br />
<br />
<h3>Encapsulation</h3><br />
<ol><br />
<li>The encapsulator was equipped with a 200-µm nozzle. </li><br />
<li>The reactor vessel was filled with 225 mL 100 mM CaCl2. </li><br />
<li>Cell-alginate mixture was transferred to a 20 mL syringe with a Luer lock. </li><br />
<li>The syringe was connected to the bead producing unit (BPU). </li><br />
<li>Microcapsules were produced at a flow rate of 12-14 units, vibration frequency 1030-1100 Hz and voltage for bead dispersion 900-1300 V. </li><br />
<li>Polymerization lasted for 10 minutes. </li><br />
<li>The polymerization solution was drained and 75 mL of 0,05% poly-L-lysine (PLL) solution was added. </li><br />
<li>Beads were incubated in PLL solution for 10 minutes. </li><br />
<li>The PLL solution was removed and beads were washed twice (for 1 and for 5 minutes) with 150 mL of MOPS buffer. </li><br />
<li>100 mL of 0,03% alginate was added and beads were incubated for 10 minutes. </li><br />
<li>Alginate solution was drained and beads were washed once with 150 mL of MOPS buffer for 1 minute. </li><br />
<li>150 mL of depolymerization solution was added for 10 minutes. </li><br />
<li>Depolymerization solution was removed and capsules were resuspended in 150 mL MOPS and collected in a bead collection flask. </li><br />
<li>MOPS was removed and microcapsules were transferred to T-75 with 10 mL DMEM, 10% FBS media supplemented with penicillin and streptomycin. </li><br />
</ol><br />
<br />
<br/><br />
<p><b>Buffers and solutions</b><br />
<br/><br />
<br><b>10 mM MOPS buffer</b> (pH = 7,2)<br />
<br><b>MOPS buffer with NaCl</b> (pH = 7,2): 10 mM MOPS, 0,85% NaCl<br />
<br><b>Polymerisation solution</b> (pH = 7,2): 10 mM MOPS, 100 mM CaCl2<br />
<br><b>Depolymerisation solution</b> (pH = 7,2): 10 mM MOPS, 50 mM Na3-citrate, 0,45% NaCl<br />
<br><b>0,05% poly-L-lysine</b> (15-30 kDa) in MOPS buffer (pH = 7,3) <br />
<br><b>0,03% alginate</b> in MOPS buffer (pH = 7,2)<br />
<br><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2)</p><br />
</br><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="proteindetection"> Protein detection </a></h2><br />
<h3>SDS-PAGE</h3><br />
<ol><br />
<li>Samples were loaded on a 12% acrylamide gel and ran at a constant voltage (200 V) for 1 h. </li><br />
<li>Proteins were then blotted on a nitrocellulose membrane at a constant current (350 mA) for 1 h. </li><br />
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li><br />
</ol><br />
</br><br />
<br />
<h3>Immunodetection </h3><br />
<ol><br />
<li>All proteins to be detected had a Myc tag at the C-terminus. </li><br />
<li>The membrane was incubated with primary antibodies (rabbit anti-Myc diluted 1:500) overnight at 4 °C and 150 rpm. Membrane was washed. </li><br />
<li>Washing in wash buffer three times for 5 minutes each.</li><br />
<li>Membrane was incubated with secondary antibodies (anti-rabbit secondary antibodies, conjugated with HRP, diluted 1:3000) for 45 minutes at room temperature and 150 rpm. </li><br />
<li>HRP activity was detected by addition of SuperSignal West Femto or Pico Substrate (Thermo Scientific). Images were captured with Syngene G:Box chemiluminescent imaging system. </li><br />
</ol><br />
<p><b>Buffers and solutions </b></p><br />
<p><b>Wash buffer</b>: 1x PBS, 0,01% (v/v) Tween 20</p><br />
</br><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Clackson, T. (2000) Regulated gene expression systems. <i> Gene ther.</i> <b>7</b>, 120–125.<br/><br/><br />
Deuschle, U., Meyer, W.K. and Thiesen, R. (1995) Tetracycline-reversible silencing of eukaryotic promoters. <i>Mol. Cell. Biol. </i> <b>15</b>, 1907–1914.<br/><br/><br />
Kramer, B.P., Fischer, C. and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells. <i> Biotech. Bioeng.</i> <b>87</b>, 478–484.<br/><br/><br />
Pollock, R. and Clackson, T. (2002) Dimerizer-regulated gene expression. <i> Curr. Opin. Biotech. </i> <b>13</b>, 459–467.<br/><br/><br />
Gibson, D.G., Young, L., Chuang, R., Venter J.C., Hutchison III, C. A. and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature methods.</i> <b>6</b>, 343–345.<br />
</p><br />
<br />
<br />
<br/><br />
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Dusanv
http://2012.igem.org/File:Svn12_methods_fig1.PNG
File:Svn12 methods fig1.PNG
2012-10-26T17:39:22Z
<p>Dusanv: </p>
<hr />
<div></div>
Dusanv
http://2012.igem.org/Team:Slovenia/Notebook
Team:Slovenia/Notebook
2012-10-26T17:38:15Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<br/><br />
<h1><a name="naslov"></a>Experimental methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#cloning">Cloning</a><br/></li><br />
<li><a href="#cellcultures">Cell cultures</a></li><br />
<li><a href="#inductionsystems">Induction systems</a></li><br />
<li><a href="#effectors">Effectors</a></li><br />
<li><a href="#microscopy">Microscopy</a></li><br />
<li><a href="#flowcytometry">Flow cytometry</a></li><br />
<li><a href="#microencapsulation">Microencapsulation</a></li><br />
<li><a href="#proteindetection">Protein detection</a></li><br />
</ul><br />
</p><br />
<br />
<br />
<br />
<br />
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<img style="float:left; margin-right:0px;" src="https://static.igem.org/mediawiki/2012/a/a7/SVN12_7_notebook_cloning.png"/><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SVN12_7_notebook_cell_cultures.png"/><br />
<div style="clear:both"></div><br />
</td><br />
</tr><br />
<br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 1.</b> Schematic presentation of methods used for cloning and culturing eukaryotic cells.<br />
</td><br />
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<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/8/84/SVN12_7_notebook_switch.png"/></td><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/e/e7/SVN12_7_notebook_safety_mech.png"/></td><br />
</tr><br />
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<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/9/91/SVN12_7_notebook_encapsulation.png"/></td><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/c/c8/SVN12_7_notebook_effectors.png"/></td><br />
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<table class="invisible" style="width:80%;"><br />
<tbody><br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 2.</b> Schematic presentation of methods used for characterizing the switch, safety mechanisms, microencapsulation and effectors.</td><br />
</tr><br />
</tbody><br />
</table><br />
<br />
<h2><a name="cloning"> Cloning </a></h2><br />
<h3>Plasmid DNA isolation</h3><br />
<p><b>MINI PREPs for analysis and sequencing</b></p><br />
<ol><br />
<li>A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). </li><br />
<li>Bacteria were grown over night at 37 °C with agitation. </li><br />
<li>Plasmid DNA was isolated from 6-10 mL of over-night culture with GeneJET plasmid miniprep kit according to the manufacturer's protocol. </li><br />
<li>Amounts ranging from 6-10 µg of plasmid DNA were obtained. </li><br />
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Fragment DNA isolation from agarose gel</h3><br />
<p><b>AGAROSE ELECTROPHORESIS</b></p><br />
<ol><br />
<li>A mixture of different sized DNA fragments was separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li><br />
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li><br />
</ol><br />
</br><br />
<p><b>FRAGMENT ISOLATION from agarose gel </b></p><br />
<ol><br />
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li><br />
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li><br />
<li>Purity and amount of DNA was determined using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Restriction digest</h3><br />
<ol><br />
<li>To digest the desired DNA restriction reactions were prepared as follows: </li><br />
<ul style="margin-left:15px;"> <b>for analysis of cloned DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>0.5 µL restriction enzyme</li><br />
<br />
<li>Bring volume to 20 µL with nuclease-free water.</li><br />
</ul><br />
<i>or</i><br />
<ul style="margin-left:15px;"> <b>for isolation of specific DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>up to 2 µL restriction enzyme </li><br />
<br />
<li>Bring volume to 50 µL with nuclease-free water.</li><br />
</ul><br />
</li><br />
<li>The sample was incubated at optimal temperature for the restriction enzymes.</li><br />
<li>Analysis of fragmented DNA was done by gel electrophoreses. </li><br />
<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<h3>PCR reaction</h3><br />
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p><br />
<ol><br />
<br />
<li>The master mix for reactions with Phusion DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (1-10 ng)</li><br />
<br />
<li>both primers (0,4 pmol/µl )</li><br />
<br />
<li>1x Phusion HF buffer</li><br />
<br />
<li>0,2 µM dNTPs</li><br />
<br />
<li>Phusion polymerase (0,02 U/ µl) and </li><br />
<br />
<li>MQ up to final volume of 25 µl</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with AccuPrime DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (10 ng),</li><br />
<br />
<li>both primers (0,4 pmol/µl ),</li><br />
<br />
<li>1xRnx mix,</li><br />
<br />
<li>enzyme (0,05 U/ µl) and</li><br />
<br />
<li>MQ up to final volume of 50 µl.</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with Taq DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>both primers (0,4 pmol/µl),</li><br />
<br />
<li>1x Taq PCR buffer II,</li><br />
<br />
<li>0,2 µM dNTPs,</li><br />
<br />
<li>5mM MgSO4,</li><br />
<br />
<li>enzyme (0,125 U/ µl) and</li><br />
<br />
<li>MQ up to total volume of 20 µl.</li><br />
<br />
<li>Then the bacterial colony was added to the reaction mix.</li> <br />
</ul><br />
</li><br />
<br />
<li>All temperature profiles were optimized according to manufacturer’s protocol,the melting temperature of primers, and the length of the desired PCR products. Reactions were performed in the Applied Biosystems Veriti 96 well thermal cycler. </li><br />
</ol><br />
</br><br />
<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p><br />
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p><br />
<h3>Gibson assembly </h3><br />
<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p><br />
<ol><br />
<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li><br />
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Ligation</h3><br />
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p><br />
<ol><br />
<li>Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA). </li><br />
<li>A ligation mixture was prepared: </li><br />
<br/><br />
1X ligase buffer (10X)<br />
<br/><br />
1 µL T4 ligase (3 U/µL)<br />
<br/><br />
Bring volume to 10 or 20 µL with nuclease-free water.<br />
</li></br><br />
<i>or</i><br />
<br />
<li>Blunt-end ligation reactions were incubated at 17 °C for 4 to 18 hours. </li><br />
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li><br />
</ol><br />
</br><br />
<br />
<h3> Culturing bacteria</h3><br />
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p><br />
<p><b> Growth media for bacteria</b></p><br />
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p><br />
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p><br />
</br><br />
<br />
<h3>Transformation of bacteria</h3><br />
<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p><br />
<ol><br />
<li>100 µL of competent cells were thawed on ice. </li><br />
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li><br />
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li><br />
<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li><br />
<li>Cooled for 3 minutes on ice. </li><br />
<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li><br />
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li><br />
</ol><br />
</br><br />
<br />
<h3>Glycerol stock for long term storage of bacteria</h3><br />
<ol><br />
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li><br />
<li>Mixed and incubated at room temperature for 30 minutes. </li><br />
<li>Afterwards the glycerol stock was stored at -80 °C. </li><br />
</ol><br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="cellcultures"> Cell cultures </a></h2><br />
<h3>Eucaryotic cell lines and cultivation</h3><br />
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p><br />
</br><br />
<br />
<h3>Subculturing monolayer cell cultures</h3><br />
<ol><br />
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li><br />
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li><br />
<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li><br />
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li><br />
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li><br />
<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li><br />
<li>Remove the trypsin-containing medium from the centrifuge tube. </li><br />
<li>Resuspend the cell pellet in fresh medium. </li><br />
<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li><br />
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li><br />
</ol><br />
</br><br />
<h3>Cell plating</h3><br />
<br />
<ol><br />
<li>Count cells. </li><br />
<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li><br />
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li><br />
</ol><br />
<br/><br />
<p><b>Media and buffers</b><br />
<br/><br />
<br><b>DMEM</b> supplemented with: 1 % L-Glutamine (GlutaMax), 10 % FBS, Optionally: 1% Pen/Strep.<br />
<br><b>RPMI</b> supplemented with: 1 % L-Glutamine (GlutaMax), 20 % FBS.</p><br />
<br />
<br/><br />
<h3>Transfection</h3><br />
TABLE: Transfection mixtures for different culture format<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Culture format</th><br />
<th>jetPEI reagent per µg of DNA (µL)</th><br />
<th>Typical amount of DNA (ng)</th><br />
<th>Volume of 150 mM NaCl solution for DNA and jetPEI (µL)</th><br />
<th>Total transfection mixture volume (µL)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">96-well</td><br />
<td class="normal">2</td><br />
<td class="normal">200</td><br />
<td class="normal">10</td><br />
<td class="normal">20</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">24-well and 8-well microscope chamber</td><br />
<td class="normal">2</td><br />
<td class="normal">500</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal">12-well</td><br />
<td class="normal">2</td><br />
<td class="normal">1000</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">6-well</td><br />
<td class="normal">2</td><br />
<td class="normal">2000</td><br />
<td class="normal">100</td><br />
<td class="normal">200</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">10 cm</td><br />
<td class="normal">2</td><br />
<td class="normal">15000</td><br />
<td class="normal">250</td><br />
<td class="normal">500</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<ol><br />
<li>Dilute plasmid DNA to desired concentration in 150 mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Dilute jetPEI (PolyPlus) in 150mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Add the jetPEI solution to the DNA solution. </li><br />
<li>Vortex the solution immediately and spin down briefly. </li><br />
<li>Incubate for 15 to 30 minutes at room temperature. </li><br />
<li>Add the jetPEI/DNA mix to the cells in and gently swirl the plate. </li><br />
<li>Return the plate to a cell culture incubator. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="inductionsystems"> Induction systems </a></h2><br />
<br />
<h3>Induction systems</h3><br />
<br />
<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p><br />
<br />
<h3>Tetracycline, erythromycin and pristinamycin systems</h3><br />
<br />
<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Svn12_parts_inducibilni.png"></img><br />
<center><p><b> Figure 1: Induced expression of TAL</b>.</p></center><br />
<br />
<p>TABLE</p><br />
<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> System </th><br />
<br />
<th> Tetracycline </th><br />
<br />
<th> Erythromycin</th><br />
<br />
<th> Pristinamycin </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> Regulating protein </td><br />
<td class="normal"> TetR:KRAB </td><br />
<td class="normal"> E:KRAB </td><br />
<td class="normal"> PIP:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> TRE </td><br />
<td class="normal"> ETR </td><br />
<td class="normal"> PIR </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> Inducer </td><br />
<td class="normal"> Tetracycline or doxycycline </td><br />
<td class="normal"> Erythromycin </td><br />
<td class="normal"> Pristinamycin </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> pCMV_TRE_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_ETR_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> pCMV_TRE_TAL-B:VP16</td><br />
<td class="normal"> pCMV_ETR_TAL-B:VP16</td><br />
<td class="normal"> pCMV_PIR_TAL-A:VP16</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-B:KRAB </td><br />
<td class="normal"> pSV40_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-A:VP16 </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pSV40_ETR_TAL-B:KRAB </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<h3>Rapamycin system</h3><br />
<br />
<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p><br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> Regulating vector </th><br />
<br />
<th> HetAct </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> ZFHD </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> ZFHD_pMIN_TAL-A:KRAB </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> ZFHD_pMIN_TAL-B:VP16</td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
</br><br />
<h3>Induction of cells</h3><br />
<ol><br />
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li><br />
<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li><br />
</ol><br />
</br><br />
<br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Inductor</th><br />
<th>Stock solution (solvent)</th><br />
<th>Dilution (solvent)</th><br />
<th>Concentration in cell medium (% solvent)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">Rapalogue (AP21967)</td><br />
<td class="normal">1 mM (100% ethanol)</td><br />
<td class="normal">10 µM (1% ethanol)</td><br />
<td class="normal">1 µM (0,1% ethanol)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Doxycyclin</td><br />
<td class="normal">1 g/L (MQ)</td><br />
<td class="normal">10 mg/L (MQ)</td><br />
<td class="normal">1 mg/L (MQ)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Pristinamycin</td><br />
<td class="normal">50 g/L (100% DMSO)</td><br />
<td class="normal">20 mg/L (1% DMSO)</td><br />
<td class="normal">2 mg/L (0,1% DMSO)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Erythromycin</td><br />
<td class="normal">50 g/L (100% ethanol)</td><br />
<td class="normal">20 mg/L (1% ethanol)</td><br />
<td class="normal">2 mg/L (0,1% ethanol)</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<!-- ----------------------------------------------------------------- --><br />
</br><br/><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="effectors"> Effectors </a></h2><br />
<h3>Biological assay-anakinra </h3><br />
<ol><br />
<li>HEK293T cells, seeded in 6-well plate, were transfected with anakinra downstream of constitutive promoter. </li><br />
<li>Transfected cells were incubated for 48 h. </li><br />
<li>To detect anakinra's effect on NF-κB signalling pathway, other HEK293T cells were transfected with plasmid coding for Renilla luciferase and plasmid reporter with NF-κB-inducible firefly luciferase expression. HEK293T cells express IL-1R, so additional transfection with a receptor gene was notneeded. </li><br />
<li>After 24 h, the medium was removed from cells transfected with reporter plasmids and 90 μL of anakinra-producing cells' supernatant was added to these wells. </li><br />
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li><br />
</ol><br />
</br><br />
<br />
<h3> Biological assay-IFN-alpha </h3><br />
<ol><br />
<li>HEK293T cells transfected with eithera plasmid encoding IFN-alpha under the control of a constitutive promoter or an empty vector, and HEK293T cells transfected with the reporter vector were co-cultivated . </li><br />
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li><br />
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li><br />
<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li><br />
</ol><br />
</br><br />
<br />
<h3>ELISA for IFN-alpha</h3><br />
<ol><br />
<li>HEK293T cells where plated on 6 well plates and transfected with a plasmid coding for human IFN-alpha under the control of a constitutive promoter or a control plasmid (pcDNA3). </li><br />
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-fluorescence(The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in black 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega ) per well. </li><br />
<li>Fluorescence was measured using an automated plate reader. </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-luminescence (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two to three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li><br />
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Plate reader-absorbance (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li><br />
<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li><br />
<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C the absorbance was measured at 630 nm. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="microscopy"> Microscopy </a></h2><br />
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p><br />
</br><br />
<br />
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3><br />
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 200 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li><br />
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 nm and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell growth (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng GFP (pPCMV-GFP) (for transfection control). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a cell cultures were imaged. </li><br />
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite GFP reporter protein. Fluorescence emission was detected at 520-560 nm for GFP. Bright field images were used to visualize the number of cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell count (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates and transfected with different amounts of mGMK:TK30 (pPCMV_mGMK:TK30) as indicated in Figure legend. </li><br />
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the Figure legend. </li><br />
<li>After incubation the cells were resuspended by pipetting. </li><br />
<li>Cells suspensions were then mixed with trypan blue. </li><br />
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-detection of reporter proteins (The Switch) </h3><br />
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p><br />
<ol><br />
<li>HEK293T cells were seeded in an 8-well microscope chamber or 12-well plate and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>Images of cells expressing reporters were taken two days after transfection and then each day for 5 days. </li><br />
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-alginate degradation (Microencapsulation) </h3><br />
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation upon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p><br />
<ol><br />
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into well. </li><br />
<li>After the dye was evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added. </li><br />
<li>The microscope was set to capture images every 20 seconds. </li><br />
<li>Screenshots were collected for at least 15 minutes. </li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Microscopy-secreted alginate lyase enzymatic activity (Microencapsulation)</h3><br />
<ol><br />
<li>HEK293T cells were seeded 1×10<sup>6</sup> on 10 cm cell culture dish and grown in DMEM medium supplemented with 10 % FBS.</li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection).</li><br />
<li>Protein production lasted for 72 hours.</li><br />
<li>Cell supernatants were collected and concentrated 50-times using Sartorius Vivaspin 6 concentrators.</li><br />
<li>Alginate beads were produced with Büchi BIOTECH Encapsulator (see Microencapsulation: Encapsulation procedure 1.-6.).</li><br />
<li>Beads were incubated with concentrated supernatants for 72 hours in an 8-well microscope chamber.</li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into wells.</li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC detection. Fluorescence emission was detected at 520-560 nm.</li><br />
<li>Beads' diameters were assessed using Leica LAS Image Analysis software.</li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3><br />
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both live and dead cells, while 7-AAD stains dead cells only.</p><br />
<ol><br />
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS. </li><br />
<li>200 µL of the microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber. </li><br />
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added to one well. </li><br />
<li>Encapsulated cells were protected from direct light and stained for 30 minutes at 37 °C. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 543-nm line of HeNe laser was used to excite 7-AAD. </li><br />
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="flowcytometry"> Flow cytometry </a></h2><br />
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p><br />
</br><br />
<br />
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labeled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with phosphatidylserine exposed on their surface.</p><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates. </li><br />
<li>Cells were transfected with pCMV-mGMK_TK30 and treated with ganciclovir. Concentrations of ganciclovir and plasmids are indicated in Figure legends. </li><br />
<li>After incubation the cells were washed with PBS buffer and resuspended by pipetting. </li><br />
<li>Cells were pelleted with centrifugation at 1200 rpm. </li><br />
<li>The cell pellet was washed in 1x Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). </li><br />
<li>The pellet was then resuspended in 1x Annexin Binding Buffer and PE-Annexin V (5 µl per 100 μl cell suspension) was added. </li><br />
<li>Samples were incubated for 20 minutes in the dark at room temperature and then immediately analyzed with a flow cytometer. </li><br />
<li>Along with site and forward scatter, the signal in the FL2 channel (540-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p><br />
<ol><br />
<li>HEK293T cells seeded in 12-well plates were transfected with plasmids expressing MICA (pPCMV-MICA_pcDNA3).</li><br />
<li>Two days after transfection with CMV-MICA_pcDNA3, cells were resuspended in PBS at final concentration 1x10<sup>6</sup> cells/mL, stained with CFSE (0,6μM) and incubated for 10 minutes at 37°C. Staining was quenched by the addition of 5 volumes of ice-cold culture media (RPMI+ 20% FBS) to the cells. After 5 minutes incubation on ice, cells were pelleted by centrifugation and then washed by resuspending the pellet in fresh media (RPMI+ 20% FBS) a further two times for a total of three washes. CFSE was used to discriminate between HEK293T and NK-92 cells or between NK target cells K562, which were used as a positive control, and NK-92 cells.</li><br />
<li>HEK293T cells or K562 cells were mixed with NK-92 cells in different ratios (1:1, 1:5, 1:10) and incubated for 4 hours at 37 °C in culture medium consisting of RPMI, 20% FBS and hIL-2 (100 U/ml).</li><br />
<li>After incubation of HEK293T or K562 cells with NK-92 cells, cells were treated with propidium iodide.</li><br />
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded.</li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3><br />
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p><br />
<ol><br />
<li>HEK293T cells were seeded in a 12 or 24-well plate and transfected with plasmids encoding the switch. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. </li><br />
<li>Medium with inducer or without the inducer was changed after two days of cultivation. </li><br />
<li>Cells were collected at different time points (2 days after induction and then 3 days after the second media change). </li><br />
<li>Cells were washed and resuspended in PBS buffer. </li><br />
<li>A 405 nm diode laser was used to excite tagBFP and a 488-nm diode laser was used for mCitirne. </li><br />
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="microencapsulation"> Microencapsulation </a></h2><br />
<h3>Cell preparation for encapsulation</h3><br />
<ol><br />
<li>HEK 293T cells were seeded 5x10⁵ per 10 cm cell culture dish (3 per encapsulation) and grown in DMEM medium supplemented with 10 % FBS. </li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection). </li><br />
<li>The medium was removed the next day.. Transfected cells were detached using 3 mL of trypsin solution and centrifuged after the addition of fresh medium to inactivate the trypsin. </li><br />
<li>Supernatant was removed and cells were resuspended in 15 mL DMEM with 10% FBS. </li><br />
<li>Cells were counted using Countess automated cell counter (Invitrogen). </li><br />
<li>HEK 293T cells were again centrifuged and supernatant was removed. </li><br />
<li>Cells were resuspended in 2 mL of pre-warmed MOPS buffer. </li><br />
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li><br />
</ol><br />
</br><br />
<br />
<h3>Encapsulation</h3><br />
<ol><br />
<li>The encapsulator was equipped with a 200-µm nozzle. </li><br />
<li>The reactor vessel was filled with 225 mL 100 mM CaCl2. </li><br />
<li>Cell-alginate mixture was transferred to a 20 mL syringe with a Luer lock. </li><br />
<li>The syringe was connected to the bead producing unit (BPU). </li><br />
<li>Microcapsules were produced at a flow rate of 12-14 units, vibration frequency 1030-1100 Hz and voltage for bead dispersion 900-1300 V. </li><br />
<li>Polymerization lasted for 10 minutes. </li><br />
<li>The polymerization solution was drained and 75 mL of 0,05% poly-L-lysine (PLL) solution was added. </li><br />
<li>Beads were incubated in PLL solution for 10 minutes. </li><br />
<li>The PLL solution was removed and beads were washed twice (for 1 and for 5 minutes) with 150 mL of MOPS buffer. </li><br />
<li>100 mL of 0,03% alginate was added and beads were incubated for 10 minutes. </li><br />
<li>Alginate solution was drained and beads were washed once with 150 mL of MOPS buffer for 1 minute. </li><br />
<li>150 mL of depolymerization solution was added for 10 minutes. </li><br />
<li>Depolymerization solution was removed and capsules were resuspended in 150 mL MOPS and collected in a bead collection flask. </li><br />
<li>MOPS was removed and microcapsules were transferred to T-75 with 10 mL DMEM, 10% FBS media supplemented with penicillin and streptomycin. </li><br />
</ol><br />
<br />
<br/><br />
<p><b>Buffers and solutions</b><br />
<br/><br />
<br><b>10 mM MOPS buffer</b> (pH = 7,2)<br />
<br><b>MOPS buffer with NaCl</b> (pH = 7,2): 10 mM MOPS, 0,85% NaCl<br />
<br><b>Polymerisation solution</b> (pH = 7,2): 10 mM MOPS, 100 mM CaCl2<br />
<br><b>Depolymerisation solution</b> (pH = 7,2): 10 mM MOPS, 50 mM Na3-citrate, 0,45% NaCl<br />
<br><b>0,05% poly-L-lysine</b> (15-30 kDa) in MOPS buffer (pH = 7,3) <br />
<br><b>0,03% alginate</b> in MOPS buffer (pH = 7,2)<br />
<br><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2)</p><br />
</br><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="proteindetection"> Protein detection </a></h2><br />
<h3>SDS-PAGE</h3><br />
<ol><br />
<li>Samples were loaded on a 12% acrylamide gel and ran at a constant voltage (200 V) for 1 h. </li><br />
<li>Proteins were then blotted on a nitrocellulose membrane at a constant current (350 mA) for 1 h. </li><br />
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li><br />
</ol><br />
</br><br />
<br />
<h3>Immunodetection </h3><br />
<ol><br />
<li>All proteins to be detected had a Myc tag at the C-terminus. </li><br />
<li>The membrane was incubated with primary antibodies (rabbit anti-Myc diluted 1:500) overnight at 4 °C and 150 rpm. Membrane was washed. </li><br />
<li>Washing in wash buffer three times for 5 minutes each.</li><br />
<li>Membrane was incubated with secondary antibodies (anti-rabbit secondary antibodies, conjugated with HRP, diluted 1:3000) for 45 minutes at room temperature and 150 rpm. </li><br />
<li>HRP activity was detected by addition of SuperSignal West Femto or Pico Substrate (Thermo Scientific). Images were captured with Syngene G:Box chemiluminescent imaging system. </li><br />
</ol><br />
<p><b>Buffers and solutions </b></p><br />
<p><b>Wash buffer</b>: 1x PBS, 0,01% (v/v) Tween 20</p><br />
</br><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Clackson, T. (2000) Regulated gene expression systems. <i> Gene ther.</i> <b>7</b>, 120–125.<br/><br/><br />
Deuschle, U., Meyer, W.K. and Thiesen, R. (1995) Tetracycline-reversible silencing of eukaryotic promoters. <i>Mol. Cell. Biol. </i> <b>15</b>, 1907–1914.<br/><br/><br />
Kramer, B.P., Fischer, C. and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells. <i> Biotech. Bioeng.</i> <b>87</b>, 478–484.<br/><br/><br />
Pollock, R. and Clackson, T. (2002) Dimerizer-regulated gene expression. <i> Curr. Opin. Biotech. </i> <b>13</b>, 459–467.<br/><br/><br />
Gibson, D.G., Young, L., Chuang, R., Venter J.C., Hutchison III, C. A. and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature methods.</i> <b>6</b>, 343–345.<br />
</p><br />
<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/Notebook
Team:Slovenia/Notebook
2012-10-26T17:33:59Z
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
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<br />
<br />
<br/><br />
<h1><a name="naslov"></a>Experimental methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#cloning">Cloning</a><br/></li><br />
<li><a href="#cellcultures">Cell cultures</a></li><br />
<li><a href="#inductionsystems">Induction systems</a></li><br />
<li><a href="#effectors">Effectors</a></li><br />
<li><a href="#microscopy">Microscopy</a></li><br />
<li><a href="#flowcytometry">Flow cytometry</a></li><br />
<li><a href="#microencapsulation">Microencapsulation</a></li><br />
<li><a href="#proteindetection">Protein detection</a></li><br />
</ul><br />
</p><br />
<br />
<br />
<br />
<br />
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<td class="invisible"><br />
<img style="float:left;" src="https://static.igem.org/mediawiki/2012/a/a7/SVN12_7_notebook_cloning.png"/><br />
<img stlye="float:left;" src="https://static.igem.org/mediawiki/2012/8/8e/SVN12_7_notebook_cell_cultures.png"/><br />
<div style="clear:both"></div><br />
</td><br />
</tr><br />
<br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 1.</b> Schematic presentation of methods used for cloning and culturing eukaryotic cells.<br />
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<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/8/84/SVN12_7_notebook_switch.png"/></td><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/e/e7/SVN12_7_notebook_safety_mech.png"/></td><br />
</tr><br />
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<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/9/91/SVN12_7_notebook_encapsulation.png"/></td><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/c/c8/SVN12_7_notebook_effectors.png"/></td><br />
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<table class="invisible" style="width:80%;"><br />
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<td class="invisible"><b>Figure 2.</b> Schematic presentation of methods used for characterizing the switch, safety mechanisms, microencapsulation and effectors.</td><br />
</tr><br />
</tbody><br />
</table><br />
<br />
<h2><a name="cloning"> Cloning </a></h2><br />
<h3>Plasmid DNA isolation</h3><br />
<p><b>MINI PREPs for analysis and sequencing</b></p><br />
<ol><br />
<li>A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). </li><br />
<li>Bacteria were grown over night at 37 °C with agitation. </li><br />
<li>Plasmid DNA was isolated from 6-10 mL of over-night culture with GeneJET plasmid miniprep kit according to the manufacturer's protocol. </li><br />
<li>Amounts ranging from 6-10 µg of plasmid DNA were obtained. </li><br />
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Fragment DNA isolation from agarose gel</h3><br />
<p><b>AGAROSE ELECTROPHORESIS</b></p><br />
<ol><br />
<li>A mixture of different sized DNA fragments was separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li><br />
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li><br />
</ol><br />
</br><br />
<p><b>FRAGMENT ISOLATION from agarose gel </b></p><br />
<ol><br />
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li><br />
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li><br />
<li>Purity and amount of DNA was determined using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Restriction digest</h3><br />
<ol><br />
<li>To digest the desired DNA restriction reactions were prepared as follows: </li><br />
<ul style="margin-left:15px;"> <b>for analysis of cloned DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>0.5 µL restriction enzyme</li><br />
<br />
<li>Bring volume to 20 µL with nuclease-free water.</li><br />
</ul><br />
<i>or</i><br />
<ul style="margin-left:15px;"> <b>for isolation of specific DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>up to 2 µL restriction enzyme </li><br />
<br />
<li>Bring volume to 50 µL with nuclease-free water.</li><br />
</ul><br />
</li><br />
<li>The sample was incubated at optimal temperature for the restriction enzymes.</li><br />
<li>Analysis of fragmented DNA was done by gel electrophoreses. </li><br />
<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<h3>PCR reaction</h3><br />
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p><br />
<ol><br />
<br />
<li>The master mix for reactions with Phusion DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (1-10 ng)</li><br />
<br />
<li>both primers (0,4 pmol/µl )</li><br />
<br />
<li>1x Phusion HF buffer</li><br />
<br />
<li>0,2 µM dNTPs</li><br />
<br />
<li>Phusion polymerase (0,02 U/ µl) and </li><br />
<br />
<li>MQ up to final volume of 25 µl</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with AccuPrime DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (10 ng),</li><br />
<br />
<li>both primers (0,4 pmol/µl ),</li><br />
<br />
<li>1xRnx mix,</li><br />
<br />
<li>enzyme (0,05 U/ µl) and</li><br />
<br />
<li>MQ up to final volume of 50 µl.</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with Taq DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>both primers (0,4 pmol/µl),</li><br />
<br />
<li>1x Taq PCR buffer II,</li><br />
<br />
<li>0,2 µM dNTPs,</li><br />
<br />
<li>5mM MgSO4,</li><br />
<br />
<li>enzyme (0,125 U/ µl) and</li><br />
<br />
<li>MQ up to total volume of 20 µl.</li><br />
<br />
<li>Then the bacterial colony was added to the reaction mix.</li> <br />
</ul><br />
</li><br />
<br />
<li>All temperature profiles were optimized according to manufacturer’s protocol,the melting temperature of primers, and the length of the desired PCR products. Reactions were performed in the Applied Biosystems Veriti 96 well thermal cycler. </li><br />
</ol><br />
</br><br />
<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p><br />
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p><br />
<h3>Gibson assembly </h3><br />
<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p><br />
<ol><br />
<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li><br />
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Ligation</h3><br />
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p><br />
<ol><br />
<li>Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA). </li><br />
<li>A ligation mixture was prepared: </li><br />
<br/><br />
1X ligase buffer (10X)<br />
<br/><br />
1 µL T4 ligase (3 U/µL)<br />
<br/><br />
Bring volume to 10 or 20 µL with nuclease-free water.<br />
</li></br><br />
<i>or</i><br />
<br />
<li>Blunt-end ligation reactions were incubated at 17 °C for 4 to 18 hours. </li><br />
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li><br />
</ol><br />
</br><br />
<br />
<h3> Culturing bacteria</h3><br />
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p><br />
<p><b> Growth media for bacteria</b></p><br />
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p><br />
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p><br />
</br><br />
<br />
<h3>Transformation of bacteria</h3><br />
<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p><br />
<ol><br />
<li>100 µL of competent cells were thawed on ice. </li><br />
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li><br />
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li><br />
<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li><br />
<li>Cooled for 3 minutes on ice. </li><br />
<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li><br />
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li><br />
</ol><br />
</br><br />
<br />
<h3>Glycerol stock for long term storage of bacteria</h3><br />
<ol><br />
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li><br />
<li>Mixed and incubated at room temperature for 30 minutes. </li><br />
<li>Afterwards the glycerol stock was stored at -80 °C. </li><br />
</ol><br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="cellcultures"> Cell cultures </a></h2><br />
<h3>Eucaryotic cell lines and cultivation</h3><br />
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p><br />
</br><br />
<br />
<h3>Subculturing monolayer cell cultures</h3><br />
<ol><br />
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li><br />
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li><br />
<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li><br />
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li><br />
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li><br />
<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li><br />
<li>Remove the trypsin-containing medium from the centrifuge tube. </li><br />
<li>Resuspend the cell pellet in fresh medium. </li><br />
<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li><br />
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li><br />
</ol><br />
</br><br />
<h3>Cell plating</h3><br />
<br />
<ol><br />
<li>Count cells. </li><br />
<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li><br />
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li><br />
</ol><br />
<br/><br />
<p><b>Media and buffers</b><br />
<br/><br />
<br><b>DMEM</b> supplemented with: 1 % L-Glutamine (GlutaMax), 10 % FBS, Optionally: 1% Pen/Strep.<br />
<br><b>RPMI</b> supplemented with: 1 % L-Glutamine (GlutaMax), 20 % FBS.</p><br />
<br />
<br/><br />
<h3>Transfection</h3><br />
TABLE: Transfection mixtures for different culture format<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Culture format</th><br />
<th>jetPEI reagent per µg of DNA (µL)</th><br />
<th>Typical amount of DNA (ng)</th><br />
<th>Volume of 150 mM NaCl solution for DNA and jetPEI (µL)</th><br />
<th>Total transfection mixture volume (µL)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">96-well</td><br />
<td class="normal">2</td><br />
<td class="normal">200</td><br />
<td class="normal">10</td><br />
<td class="normal">20</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">24-well and 8-well microscope chamber</td><br />
<td class="normal">2</td><br />
<td class="normal">500</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal">12-well</td><br />
<td class="normal">2</td><br />
<td class="normal">1000</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">6-well</td><br />
<td class="normal">2</td><br />
<td class="normal">2000</td><br />
<td class="normal">100</td><br />
<td class="normal">200</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">10 cm</td><br />
<td class="normal">2</td><br />
<td class="normal">15000</td><br />
<td class="normal">250</td><br />
<td class="normal">500</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<ol><br />
<li>Dilute plasmid DNA to desired concentration in 150 mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Dilute jetPEI (PolyPlus) in 150mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Add the jetPEI solution to the DNA solution. </li><br />
<li>Vortex the solution immediately and spin down briefly. </li><br />
<li>Incubate for 15 to 30 minutes at room temperature. </li><br />
<li>Add the jetPEI/DNA mix to the cells in and gently swirl the plate. </li><br />
<li>Return the plate to a cell culture incubator. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="inductionsystems"> Induction systems </a></h2><br />
<br />
<h3>Induction systems</h3><br />
<br />
<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p><br />
<br />
<h3>Tetracycline, erythromycin and pristinamycin systems</h3><br />
<br />
<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Svn12_parts_inducibilni.png"></img><br />
<center><p><b> Figure 1: Induced expression of TAL</b>.</p></center><br />
<br />
<p>TABLE</p><br />
<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> System </th><br />
<br />
<th> Tetracycline </th><br />
<br />
<th> Erythromycin</th><br />
<br />
<th> Pristinamycin </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> Regulating protein </td><br />
<td class="normal"> TetR:KRAB </td><br />
<td class="normal"> E:KRAB </td><br />
<td class="normal"> PIP:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> TRE </td><br />
<td class="normal"> ETR </td><br />
<td class="normal"> PIR </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> Inducer </td><br />
<td class="normal"> Tetracycline or doxycycline </td><br />
<td class="normal"> Erythromycin </td><br />
<td class="normal"> Pristinamycin </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> pCMV_TRE_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_ETR_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> pCMV_TRE_TAL-B:VP16</td><br />
<td class="normal"> pCMV_ETR_TAL-B:VP16</td><br />
<td class="normal"> pCMV_PIR_TAL-A:VP16</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-B:KRAB </td><br />
<td class="normal"> pSV40_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-A:VP16 </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pSV40_ETR_TAL-B:KRAB </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<h3>Rapamycin system</h3><br />
<br />
<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p><br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> Regulating vector </th><br />
<br />
<th> HetAct </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> ZFHD </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> ZFHD_pMIN_TAL-A:KRAB </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> ZFHD_pMIN_TAL-B:VP16</td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
</br><br />
<h3>Induction of cells</h3><br />
<ol><br />
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li><br />
<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li><br />
</ol><br />
</br><br />
<br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Inductor</th><br />
<th>Stock solution (solvent)</th><br />
<th>Dilution (solvent)</th><br />
<th>Concentration in cell medium (% solvent)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">Rapalogue (AP21967)</td><br />
<td class="normal">1 mM (100% ethanol)</td><br />
<td class="normal">10 µM (1% ethanol)</td><br />
<td class="normal">1 µM (0,1% ethanol)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Doxycyclin</td><br />
<td class="normal">1 g/L (MQ)</td><br />
<td class="normal">10 mg/L (MQ)</td><br />
<td class="normal">1 mg/L (MQ)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Pristinamycin</td><br />
<td class="normal">50 g/L (100% DMSO)</td><br />
<td class="normal">20 mg/L (1% DMSO)</td><br />
<td class="normal">2 mg/L (0,1% DMSO)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Erythromycin</td><br />
<td class="normal">50 g/L (100% ethanol)</td><br />
<td class="normal">20 mg/L (1% ethanol)</td><br />
<td class="normal">2 mg/L (0,1% ethanol)</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<!-- ----------------------------------------------------------------- --><br />
</br><br/><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="effectors"> Effectors </a></h2><br />
<h3>Biological assay-anakinra </h3><br />
<ol><br />
<li>HEK293T cells, seeded in 6-well plate, were transfected with anakinra downstream of constitutive promoter. </li><br />
<li>Transfected cells were incubated for 48 h. </li><br />
<li>To detect anakinra's effect on NF-κB signalling pathway, other HEK293T cells were transfected with plasmid coding for Renilla luciferase and plasmid reporter with NF-κB-inducible firefly luciferase expression. HEK293T cells express IL-1R, so additional transfection with a receptor gene was notneeded. </li><br />
<li>After 24 h, the medium was removed from cells transfected with reporter plasmids and 90 μL of anakinra-producing cells' supernatant was added to these wells. </li><br />
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li><br />
</ol><br />
</br><br />
<br />
<h3> Biological assay-IFN-alpha </h3><br />
<ol><br />
<li>HEK293T cells transfected with eithera plasmid encoding IFN-alpha under the control of a constitutive promoter or an empty vector, and HEK293T cells transfected with the reporter vector were co-cultivated . </li><br />
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li><br />
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li><br />
<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li><br />
</ol><br />
</br><br />
<br />
<h3>ELISA for IFN-alpha</h3><br />
<ol><br />
<li>HEK293T cells where plated on 6 well plates and transfected with a plasmid coding for human IFN-alpha under the control of a constitutive promoter or a control plasmid (pcDNA3). </li><br />
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-fluorescence(The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in black 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega ) per well. </li><br />
<li>Fluorescence was measured using an automated plate reader. </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-luminescence (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two to three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li><br />
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Plate reader-absorbance (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li><br />
<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li><br />
<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C the absorbance was measured at 630 nm. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="microscopy"> Microscopy </a></h2><br />
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p><br />
</br><br />
<br />
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3><br />
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 200 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li><br />
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 nm and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell growth (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng GFP (pPCMV-GFP) (for transfection control). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a cell cultures were imaged. </li><br />
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite GFP reporter protein. Fluorescence emission was detected at 520-560 nm for GFP. Bright field images were used to visualize the number of cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell count (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates and transfected with different amounts of mGMK:TK30 (pPCMV_mGMK:TK30) as indicated in Figure legend. </li><br />
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the Figure legend. </li><br />
<li>After incubation the cells were resuspended by pipetting. </li><br />
<li>Cells suspensions were then mixed with trypan blue. </li><br />
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-detection of reporter proteins (The Switch) </h3><br />
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p><br />
<ol><br />
<li>HEK293T cells were seeded in an 8-well microscope chamber or 12-well plate and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>Images of cells expressing reporters were taken two days after transfection and then each day for 5 days. </li><br />
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-alginate degradation (Microencapsulation) </h3><br />
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation upon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p><br />
<ol><br />
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into well. </li><br />
<li>After the dye was evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added. </li><br />
<li>The microscope was set to capture images every 20 seconds. </li><br />
<li>Screenshots were collected for at least 15 minutes. </li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Microscopy-secreted alginate lyase enzymatic activity (Microencapsulation)</h3><br />
<ol><br />
<li>HEK293T cells were seeded 1×10<sup>6</sup> on 10 cm cell culture dish and grown in DMEM medium supplemented with 10 % FBS.</li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection).</li><br />
<li>Protein production lasted for 72 hours.</li><br />
<li>Cell supernatants were collected and concentrated 50-times using Sartorius Vivaspin 6 concentrators.</li><br />
<li>Alginate beads were produced with Büchi BIOTECH Encapsulator (see Microencapsulation: Encapsulation procedure 1.-6.).</li><br />
<li>Beads were incubated with concentrated supernatants for 72 hours in an 8-well microscope chamber.</li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into wells.</li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC detection. Fluorescence emission was detected at 520-560 nm.</li><br />
<li>Beads' diameters were assessed using Leica LAS Image Analysis software.</li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3><br />
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both live and dead cells, while 7-AAD stains dead cells only.</p><br />
<ol><br />
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS. </li><br />
<li>200 µL of the microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber. </li><br />
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added to one well. </li><br />
<li>Encapsulated cells were protected from direct light and stained for 30 minutes at 37 °C. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 543-nm line of HeNe laser was used to excite 7-AAD. </li><br />
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="flowcytometry"> Flow cytometry </a></h2><br />
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p><br />
</br><br />
<br />
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labeled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with phosphatidylserine exposed on their surface.</p><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates. </li><br />
<li>Cells were transfected with pCMV-mGMK_TK30 and treated with ganciclovir. Concentrations of ganciclovir and plasmids are indicated in Figure legends. </li><br />
<li>After incubation the cells were washed with PBS buffer and resuspended by pipetting. </li><br />
<li>Cells were pelleted with centrifugation at 1200 rpm. </li><br />
<li>The cell pellet was washed in 1x Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). </li><br />
<li>The pellet was then resuspended in 1x Annexin Binding Buffer and PE-Annexin V (5 µl per 100 μl cell suspension) was added. </li><br />
<li>Samples were incubated for 20 minutes in the dark at room temperature and then immediately analyzed with a flow cytometer. </li><br />
<li>Along with site and forward scatter, the signal in the FL2 channel (540-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p><br />
<ol><br />
<li>HEK293T cells seeded in 12-well plates were transfected with plasmids expressing MICA (pPCMV-MICA_pcDNA3).</li><br />
<li>Two days after transfection with CMV-MICA_pcDNA3, cells were resuspended in PBS at final concentration 1x10<sup>6</sup> cells/mL, stained with CFSE (0,6μM) and incubated for 10 minutes at 37°C. Staining was quenched by the addition of 5 volumes of ice-cold culture media (RPMI+ 20% FBS) to the cells. After 5 minutes incubation on ice, cells were pelleted by centrifugation and then washed by resuspending the pellet in fresh media (RPMI+ 20% FBS) a further two times for a total of three washes. CFSE was used to discriminate between HEK293T and NK-92 cells or between NK target cells K562, which were used as a positive control, and NK-92 cells.</li><br />
<li>HEK293T cells or K562 cells were mixed with NK-92 cells in different ratios (1:1, 1:5, 1:10) and incubated for 4 hours at 37 °C in culture medium consisting of RPMI, 20% FBS and hIL-2 (100 U/ml).</li><br />
<li>After incubation of HEK293T or K562 cells with NK-92 cells, cells were treated with propidium iodide.</li><br />
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded.</li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3><br />
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p><br />
<ol><br />
<li>HEK293T cells were seeded in a 12 or 24-well plate and transfected with plasmids encoding the switch. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. </li><br />
<li>Medium with inducer or without the inducer was changed after two days of cultivation. </li><br />
<li>Cells were collected at different time points (2 days after induction and then 3 days after the second media change). </li><br />
<li>Cells were washed and resuspended in PBS buffer. </li><br />
<li>A 405 nm diode laser was used to excite tagBFP and a 488-nm diode laser was used for mCitirne. </li><br />
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="microencapsulation"> Microencapsulation </a></h2><br />
<h3>Cell preparation for encapsulation</h3><br />
<ol><br />
<li>HEK 293T cells were seeded 5x10⁵ per 10 cm cell culture dish (3 per encapsulation) and grown in DMEM medium supplemented with 10 % FBS. </li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection). </li><br />
<li>The medium was removed the next day.. Transfected cells were detached using 3 mL of trypsin solution and centrifuged after the addition of fresh medium to inactivate the trypsin. </li><br />
<li>Supernatant was removed and cells were resuspended in 15 mL DMEM with 10% FBS. </li><br />
<li>Cells were counted using Countess automated cell counter (Invitrogen). </li><br />
<li>HEK 293T cells were again centrifuged and supernatant was removed. </li><br />
<li>Cells were resuspended in 2 mL of pre-warmed MOPS buffer. </li><br />
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li><br />
</ol><br />
</br><br />
<br />
<h3>Encapsulation</h3><br />
<ol><br />
<li>The encapsulator was equipped with a 200-µm nozzle. </li><br />
<li>The reactor vessel was filled with 225 mL 100 mM CaCl2. </li><br />
<li>Cell-alginate mixture was transferred to a 20 mL syringe with a Luer lock. </li><br />
<li>The syringe was connected to the bead producing unit (BPU). </li><br />
<li>Microcapsules were produced at a flow rate of 12-14 units, vibration frequency 1030-1100 Hz and voltage for bead dispersion 900-1300 V. </li><br />
<li>Polymerization lasted for 10 minutes. </li><br />
<li>The polymerization solution was drained and 75 mL of 0,05% poly-L-lysine (PLL) solution was added. </li><br />
<li>Beads were incubated in PLL solution for 10 minutes. </li><br />
<li>The PLL solution was removed and beads were washed twice (for 1 and for 5 minutes) with 150 mL of MOPS buffer. </li><br />
<li>100 mL of 0,03% alginate was added and beads were incubated for 10 minutes. </li><br />
<li>Alginate solution was drained and beads were washed once with 150 mL of MOPS buffer for 1 minute. </li><br />
<li>150 mL of depolymerization solution was added for 10 minutes. </li><br />
<li>Depolymerization solution was removed and capsules were resuspended in 150 mL MOPS and collected in a bead collection flask. </li><br />
<li>MOPS was removed and microcapsules were transferred to T-75 with 10 mL DMEM, 10% FBS media supplemented with penicillin and streptomycin. </li><br />
</ol><br />
<br />
<br/><br />
<p><b>Buffers and solutions</b><br />
<br/><br />
<br><b>10 mM MOPS buffer</b> (pH = 7,2)<br />
<br><b>MOPS buffer with NaCl</b> (pH = 7,2): 10 mM MOPS, 0,85% NaCl<br />
<br><b>Polymerisation solution</b> (pH = 7,2): 10 mM MOPS, 100 mM CaCl2<br />
<br><b>Depolymerisation solution</b> (pH = 7,2): 10 mM MOPS, 50 mM Na3-citrate, 0,45% NaCl<br />
<br><b>0,05% poly-L-lysine</b> (15-30 kDa) in MOPS buffer (pH = 7,3) <br />
<br><b>0,03% alginate</b> in MOPS buffer (pH = 7,2)<br />
<br><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2)</p><br />
</br><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="proteindetection"> Protein detection </a></h2><br />
<h3>SDS-PAGE</h3><br />
<ol><br />
<li>Samples were loaded on a 12% acrylamide gel and ran at a constant voltage (200 V) for 1 h. </li><br />
<li>Proteins were then blotted on a nitrocellulose membrane at a constant current (350 mA) for 1 h. </li><br />
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li><br />
</ol><br />
</br><br />
<br />
<h3>Immunodetection </h3><br />
<ol><br />
<li>All proteins to be detected had a Myc tag at the C-terminus. </li><br />
<li>The membrane was incubated with primary antibodies (rabbit anti-Myc diluted 1:500) overnight at 4 °C and 150 rpm. Membrane was washed. </li><br />
<li>Washing in wash buffer three times for 5 minutes each.</li><br />
<li>Membrane was incubated with secondary antibodies (anti-rabbit secondary antibodies, conjugated with HRP, diluted 1:3000) for 45 minutes at room temperature and 150 rpm. </li><br />
<li>HRP activity was detected by addition of SuperSignal West Femto or Pico Substrate (Thermo Scientific). Images were captured with Syngene G:Box chemiluminescent imaging system. </li><br />
</ol><br />
<p><b>Buffers and solutions </b></p><br />
<p><b>Wash buffer</b>: 1x PBS, 0,01% (v/v) Tween 20</p><br />
</br><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Clackson, T. (2000) Regulated gene expression systems. <i> Gene ther.</i> <b>7</b>, 120–125.<br/><br/><br />
Deuschle, U., Meyer, W.K. and Thiesen, R. (1995) Tetracycline-reversible silencing of eukaryotic promoters. <i>Mol. Cell. Biol. </i> <b>15</b>, 1907–1914.<br/><br/><br />
Kramer, B.P., Fischer, C. and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells. <i> Biotech. Bioeng.</i> <b>87</b>, 478–484.<br/><br/><br />
Pollock, R. and Clackson, T. (2002) Dimerizer-regulated gene expression. <i> Curr. Opin. Biotech. </i> <b>13</b>, 459–467.<br/><br/><br />
Gibson, D.G., Young, L., Chuang, R., Venter J.C., Hutchison III, C. A. and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature methods.</i> <b>6</b>, 343–345.<br />
</p><br />
<br />
<br />
<br/><br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'>Lablog >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim
Team:Slovenia/ModelingPositiveFeedbackLoopSwitchCSim
2012-10-26T17:26:32Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<br />
<h1>Modeling - positive feedback loop switch</h1><br />
<p><br />
<ol><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch"><b>Deterministic model</b></a></li><br />
<li><a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitchStochastic"><b>Stochastic model</b></a></li><br />
<li><b>C#Sim model</b></li><br />
<ul style="margin-left:30px;"><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#model">The model</a></li><br />
<li><a href="#results">Simulation results</a></li><br />
</ul><br />
</ol><br />
</p><br />
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<br />
<br />
<br />
<h2><a name="summary">C#Sim model of the positive feedback loop switch</a></h2><br />
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<p><br />
C#Sim model of the positive feedback loop switch, like other modeling approaches, showed that this switch was much more robust than the mutual repressor switch. The positive feedback loop switch would exhibit bistability even for low transcription factor exponent values, such as 1.1 - much lower than required for bistability of the mutual repressor switch. It again proved tolerant to leaky production of transcription factors and exhibited bistability even for low translation effectiveness (e.g. 25%).<br />
</p><p><br />
Decreasing translation effectiveness required a relatively slight increase in transcription factor exponent values for bistability to occur.<br />
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<h2><a name="model">The model</a></h2><br />
<p><br />
The model was constructed in C# programming language by defining objects that represented the switch. See <a href="https://2012.igem.org/Team:Slovenia/SourceCode">source code</a> for complete implementation details. See <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">modeling methods</a> for algorithm description.<br />
</p><br />
<br />
<br />
<h2><a name="results">Simulation results</a></h2><br />
<p><br />
State-switching was achieved by introducing state-inducing signals for a certain duration of time. Each signal was modeled as a step function. Each binding site had a capacity equal to 10, to represent 10 binding site repeats. Active transcription rates (k) of all promoters were equal to 200 units. mRNA degradation percentage per simulation step was 0.25 and protein degradation percentage was 0.1.<br />
</p><br />
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<p><br />
Simulation results show reached protein levels (i.e. the amount of protein entities in the system) as a function of time.<br />
</p><br />
<br />
<p><br />
In our first test, the following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 to induce stable state 2 (high mCitrine) and removed at time = 100;</li><br />
<li>signal 1 was introduced at time = 200 to induce stable state 1 (high BFP) and removed at time = 300;</li><br />
<li>signal 2 was again introduced at time = 400 and removed at time = 500;</li><br />
<li>signal 1 was again introduced at time = 600 and removed at time = 700.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Leaky expression (b) of each gene was equal to 15 units (compared to active transcription rate of 200, that means leaking of 7,5%). Exponent values (m and n - see <a href="https://2012.igem.org/Team:Slovenia/ModelingMethods">Modeling methods</a> for description) were equal to 1.3. Translation effectiveness was 25%. While the mutual repressor switch didn't exhibit bistability for this parameter values, the positive feedback loop switch did, as shown in figure 1.<br />
</p><br />
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<b>Figure 1. </b>The positive feedback loop switch transitioning between stable states for parameter values that did not result in bistability of the mutual repressor switch (here, transcription factor exponents were equal to 1.3 and leaky transcription rates were 15 units).<br />
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For our next tests, the following state-switching scenario was used:<br />
<ul style="margin-left:30px;"><br />
<li>signal 2 was introduced at time = 0 (with time here we mean simulation step number) to induce stable state 2 (high mCitrine) and removed at <br/>time = 100;</li><br />
<li>signal 1 was introduced at time = 400 to induce stable state 1 (high BFP) and removed at time = 500;</li><br />
<li>signal 2 was again introduced at time = 800 and removed at time = 900;</li><br />
<li>signal 1 was again introduced at time = 1200 and removed at time = 1300.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Exponents were equal to 1.1. Leaky production rate of all proteins was again 15 units. Translation effectiveness was 90%. Bistability was exhibited, as shown in figure 2. Decreasing translation effectiveness required a slight increase in exponent values for bistability to occur. Figure 3 shows that bistability was exhibited fo exponent values of 1.3 when translation effectiveness was reduced to 40%. Figure 4 shows the switch exhibiting bistability for exponent values equal to 1.3 with leaky transcription rate equal to 15 units and translation effectiveness equal to 100%.<br />
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<b>Figure 2.</b> The positive feedback loop switch exhibited bistability for exponent values of 1.1 when translation effectiveness was 90%, despite leaky expression of 15 units for all genes.<br />
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<b>Figure 3.</b> The positive feedback loop switch exhibited bistability for exponent values of 1.3 when translation effectiveness was 40%.<br />
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<b>Figure 4.</b> The positive feedback loop switch exhibiting bistability for translation effectiveness of 100%.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'>Experimental model >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/Modeling
Team:Slovenia/Modeling
2012-10-26T17:25:02Z
<p>Dusanv: </p>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<h1>Modeling overview</h1><br />
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<b style="font-size:120%;">Pharmacokinetic modeling</b><br />
<p><br />
Distribution of drugs throughout tissues is very important for the effective therapy. We built <b>pharmacokinetic models</b> to simulate distribution of biological drugs produced by the engineered microencapsulated cells which would be implanted into the liver to treat hepatitis C and into the heart for the therapy of myocardial ischaemia, in comparison to the standard therapy.</p><br />
<p><br />
Pharmacokinetic models suggest that <b>the proposed type of delivery should decrease the systemic side effects and the required dose of biological drugs.</b><br />
</p><br />
</p><br />
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<br />
<br/><br/><br />
<p><br />
<b style="font-size:120%;">Modeling of epigenetic switches</b><br />
<p><br />
Mathematical modeling was used to simulate different types of epigenetic switches (mutual repressor switch, based on the classic toggle switch and its extended version with introduction of additional positive feedback loops), where experimental parameters were incorporated into the model. Our models led to some non-intuitive results concerning the introduction of non-linearity into the system, which was verified by experimental results.<br />
</p><br />
<br />
<p><br />
We constructed <b>deterministic and stochastic models</b> to analyze both of our switches and developed <b>two new modeling approaches</b>:<br />
<ul style="margin-left:30px;"><br />
<li><b><a href="https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel">an experimental model</a></b> based on the available experimental data;</li><br />
<li><b><a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">a new modeling algorithm</a></b>, called C#Sim, based on object-oriented programming approach.</li><br />
</ul><br />
</p><br />
</p><br />
<br />
<br/><br/><br />
<p><br />
<b style="font-size:120%;">What did the dry-lab analysis of epigenetic switches show?</b><br />
<p><br />
All models consistently demonstrate that:<br />
<ul style="margin-left:30px;"><br />
<li>the mutual repressor switch is unlikely to exhibit bistability in a realistic experimental setting using monomeric transcription factors;</li><br />
<li>the positive feedback loop switch is, in terms of robustness, far superior to the mutual repressor switch based on non-cooperative orthogonal DNA-binding domains of transcription factors, exhibiting bistability in more demanding (non-ideal) conditions.</li><br />
</ul><br />
</p><br />
</p><br />
<br />
<p><br />
Therefore we predicted that the mutual repressor switch would not exhibit bistable behavior, while the positive feedback loop switch should be stable. These assessments were confirmed by experimental results, with the positive feedback loop switch clearly exhibiting bistability. <br />
</p><br />
<br />
<br />
<br/><br/><br />
<p><br />
<b style="font-size:120%;">How did modeling help our project?</b><br />
<p>Pharmacokinetic model made it possible to compare conventional and our therapy and to calculate required drug production in microencapsulated cells.</p><br />
<p>Modeling of epigenetic switches and thorough parameter space analysis made it possible to provide our wet-lab with answers to questions regarding how our switches may behave in different scenarios - e.g., how different amounts of constructs defining the positive feedback loop switch may affect bistability, what is the effect of leaky transcription, can the positive feedback loops replace the need for transcription factor cooperativity to obtain bistability, why the mutual repressor switch may not work, etc. This way, our experimental work was interwoven with dry-lab modeling.</p> <br />
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<br />
<h2>Pharmacokinetic modeling</h2><br />
<p><br />
A pharmacokinetic model was built to simulate drug distribution troughout body tissues. We used physiologically based design to construct a mathematical model and predict drug kinetics. Several models were built to compare standard therapies with localized therapeutical cells.</p><p><br />
Calculations show that localized drug production accounts for better concentration ratios between target and non-target tissues and also maintains steady concentration levels through time. This has a potential to avoid many of the side effects with common therapies. In addition, with this therapy, drug concentration does not fluctuate as in standard therapy. Peaks in concentration contribute to side-effects while decreases in concentrations cause lower therapeutical effectiveness. Results suggest this prospective treatment provides a more efficient and safe alternative.<br />
<br />
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<b>Figure 1.</b> A real-time visualization of our pharmacokinetic model depicting therapeutics concentrations in various tissues.<br />
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<br />
<br />
<h2>Deterministic and stochastic modeling</h2><br />
<p>We modeled the mutual repressor switch and the positive feedback loop switch using deterministic and stochastic modeling approach. The deterministic model was based on the probabilistic interpretation of gene regulation and formalized as a set of ordinary differential equations. For each promoter, the probability of it being in an active state (i.e. a state leading to gene expression) was formulated mathematically, considering transcription factors bound to the corresponding binding sites. In this way, binding of an activator would result in activation of transcription from the minimal promoter, while binding of a repressor would result in an inactive promoter. Stochastic models were formulated as a set of reactions describing the dynamics of a switch and simulated using stochastic simulation algorithm.<br />
<br />
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<b>Figure 2.</b> The positive feedback loop switch exhibiting bistability in a stochastic simulation even in the presence of leaky transcription of designed TAL repressors and no cooperativity. The mutual repressor switch did not exhibit bistability in such conditions. Reporter concentrations are depicted as a function of time.<br />
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<br />
<br />
<h2>Experimental modeling</h2><br />
<p><br />
An experimental model of switch dynamics extracted simulation parameters from the available experimental data of gene regulation and results obtained from our experiments using TAL regulators. This model also predicted that the positive feedback loop switch exhibits bistability without cooperative DNA binding.<br />
</p><br />
<br />
<br />
<h2>C#Sim - a new object oriented hybrid modeling algorithm </h2><br />
<p><br />
We developed a new modeling algorithm, called C#Sim. This algorithm enabled us:<br />
<ul style="margin-left:30px;"><br />
<li>to explicitly model transcription factor binding, especially competitive binding of TAL activators and repressors to the same binding site, characteristic of our positive feedback loop switch;</li><br />
<li>to explicitly model a limited number of binding site repeats;</li><br />
<li>to incorporate the stochasticity of gene expression into an otherwise deterministic approach.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
The algorithm was designed in a modular, object-oriented way, allowing us to represent each mRNA and protein molecule as a separate entity with its own set of parameters. With C#Sim, gene regulatory networks can easily be constructed using a programming language and simulated as a series of related entities (i.e. objects) such as promoters, binding sites and genes. The algorithm was implemented in C# programming language (Figure 3).<br />
</p><br />
<br />
<p><br />
We modeled both the mutual repressor switch and the positive feedback loop switch using C#Sim. The results obtained led to the same conclusions as other modeling methods.<br />
</p><br />
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<b>Figure 3.</b> C#Sim algorithm implementation, displaying positive feedback loop switch simulation results.<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingPK'>Pharmacokinetic model >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/Modeling
Team:Slovenia/Modeling
2012-10-26T17:19:46Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<h1>Modeling overview</h1><br />
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<td class="summary" style="font-size:110%;"><br />
<p><br />
<b style="font-size:120%;">Pharmacokinetic modeling</b><br />
<p><br />
Distribution of drugs throughout tissues is very important for the effective therapy. We built <b>pharmacokinetic models</b> to simulate distribution of biological drugs produced by the engineered microencapsulated cells which would be implanted into the liver to treat hepatitis C and into the heart for the therapy of myocardial ischaemia, in comparison to the standard therapy.</p><br />
<p><br />
Pharmacokinetic models suggest that <b>the proposed type of delivery should decrease the systemic side effects and the required dose of biological drugs.</b><br />
</p><br />
</p><br />
<br />
<br />
<br/><br/><br />
<p><br />
<b style="font-size:120%;">Modeling of epigenetic switches</b><br />
<p><br />
Mathematical modeling was used to simulate different types of epigenetic switches (mutual repressor switch, based on the classic toggle switch and its extended version with introduction of additional positive feedback loops), where experimental parameters were incorporated into the model. Our models led to some non-intuitive results concerning the introduction of non-linearity into the system, which was verified by experimental results.<br />
</p><br />
<br />
<p><br />
We constructed <b>deterministic and stochastic models</b> to analyze both of our switches and developed <b>two new modeling approaches</b>:<br />
<ul style="margin-left:30px;"><br />
<li><b><a href="https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel">a quantitative model</a></b> based on the available experimental data;</li><br />
<li><b><a href="https://2012.igem.org/Team:Slovenia/ModelingMethods#csim">a new modeling algorithm</a></b>, called C#Sim, based on object-oriented programming approach.</li><br />
</ul><br />
</p><br />
</p><br />
<br />
<br/><br/><br />
<p><br />
<b style="font-size:120%;">What did the dry-lab analysis of epigenetic switches show?</b><br />
<p><br />
All models consistently demonstrate that:<br />
<ul style="margin-left:30px;"><br />
<li>the mutual repressor switch is unlikely to exhibit bistability in a realistic experimental setting using monomeric transcription factors;</li><br />
<li>the positive feedback loop switch is, in terms of robustness, far superior to the mutual repressor switch based on non-cooperative orthogonal DNA-binding domains of transcription factors, exhibiting bistability in more demanding (non-ideal) conditions.</li><br />
</ul><br />
</p><br />
</p><br />
<br />
<p><br />
Therefore we predicted that the mutual repressor switch would not exhibit bistable behavior, while the positive feedback loop switch should be stable. These assessments were confirmed by experimental results, with the positive feedback loop switch clearly exhibiting bistability. <br />
</p><br />
<br />
<br />
<br/><br/><br />
<p><br />
<b style="font-size:120%;">How did modeling help our project?</b><br />
<p>Pharmacokinetic model made it possible to compare conventional and our therapy and to calculate required drug production in microencapsulated cells.</p><br />
<p>Modeling of epigenetic switches and thorough parameter space analysis made it possible to provide our wet-lab with answers to questions regarding how our switches may behave in different scenarios - e.g., how different amounts of constructs defining the positive feedback loop switch may affect bistability, what is the effect of leaky transcription, can the positive feedback loops replace the need for transcription factor cooperativity to obtain bistability, why the mutual repressor switch may not work, etc. This way, our experimental work was interwoven with dry-lab modeling.</p> <br />
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<h2>Pharmacokinetic modeling</h2><br />
<p><br />
A pharmacokinetic model was built to simulate drug distribution troughout body tissues. We used physiologically based design to construct a mathematical model and predict drug kinetics. Several models were built to compare standard therapies with localized therapeutical cells.</p><p><br />
Calculations show that localized drug production accounts for better concentration ratios between target and non-target tissues and also maintains steady concentration levels through time. This has a potential to avoid many of the side effects with common therapies. In addition, with this therapy, drug concentration does not fluctuate as in standard therapy. Peaks in concentration contribute to side-effects while decreases in concentrations cause lower therapeutical effectiveness. Results suggest this prospective treatment provides a more efficient and safe alternative.<br />
<br />
<br />
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<b>Figure 1.</b> A real-time visualization of our pharmacokinetic model depicting therapeutics concentrations in various tissues.<br />
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<br />
<br />
<h2>Deterministic and stochastic modeling</h2><br />
<p>We modeled the mutual repressor switch and the positive feedback loop switch using deterministic and stochastic modeling approach. The deterministic model was based on the probabilistic interpretation of gene regulation and formalized as a set of ordinary differential equations. For each promoter, the probability of it being in an active state (i.e. a state leading to gene expression) was formulated mathematically, considering transcription factors bound to the corresponding binding sites. In this way, binding of an activator would result in activation of transcription from the minimal promoter, while binding of a repressor would result in an inactive promoter. Stochastic models were formulated as a set of reactions describing the dynamics of a switch and simulated using stochastic simulation algorithm.<br />
<br />
<br />
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<b>Figure 2.</b> The positive feedback loop switch exhibiting bistability in a stochastic simulation even in the presence of leaky transcription of designed TAL repressors and no cooperativity. The mutual repressor switch did not exhibit bistability in such conditions. Reporter concentrations are depicted as a function of time.<br />
</td></tr><br />
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<br />
<br />
<h2>Quantitative modeling</h2><br />
<p><br />
A quantitative model of switch dynamics extracted simulation parameters from the available experimental data of gene regulation and results obtained from our experiments using TAL regulators. This model also predicted that the positive feedback loop switch exhibits bistability without cooperative DNA binding.<br />
</p><br />
<br />
<br />
<h2>C#Sim - a new object oriented hybrid modeling algorithm </h2><br />
<p><br />
We developed a new modeling algorithm, called C#Sim. This algorithm enabled us:<br />
<ul style="margin-left:30px;"><br />
<li>to explicitly model transcription factor binding, especially competitive binding of TAL activators and repressors to the same binding site, characteristic of our positive feedback loop switch;</li><br />
<li>to explicitly model a limited number of binding site repeats;</li><br />
<li>to incorporate the stochasticity of gene expression into an otherwise deterministic approach.</li><br />
</ul><br />
</p><br />
<br />
<p><br />
The algorithm was designed in a modular, object-oriented way, allowing us to represent each mRNA and protein molecule as a separate entity with its own set of parameters. With C#Sim, gene regulatory networks can easily be constructed using a programming language and simulated as a series of related entities (i.e. objects) such as promoters, binding sites and genes. The algorithm was implemented in C# programming language (Figure 3).<br />
</p><br />
<br />
<p><br />
We modeled both the mutual repressor switch and the positive feedback loop switch using C#Sim. The results obtained led to the same conclusions as other modeling methods.<br />
</p><br />
<br />
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<b>Figure 3.</b> C#Sim algorithm implementation, displaying positive feedback loop switch simulation results.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/ModelingPK'>Pharmacokinetic model >></a><br />
</b><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/Notebook
Team:Slovenia/Notebook
2012-10-26T17:08:55Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<br />
<br />
<br/><br />
<h1><a name="naslov"></a>Experimental methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#cloning">Cloning</a><br/></li><br />
<li><a href="#cellcultures">Cell cultures</a></li><br />
<li><a href="#inductionsystems">Induction systems</a></li><br />
<li><a href="#effectors">Effectors</a></li><br />
<li><a href="#microscopy">Microscopy</a></li><br />
<li><a href="#flowcytometry">Flow cytometry</a></li><br />
<li><a href="#microencapsulation">Microencapsulation</a></li><br />
<li><a href="#proteindetection">Protein detection</a></li><br />
</ul><br />
</p><br />
<br />
<br />
<br />
<br />
<!-- figure 1 --><br />
<p><br />
<table class="invisible" style="width:80%;"><br />
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<td class="invisible"><br />
<img style="float:left;" src="https://static.igem.org/mediawiki/2012/a/a7/SVN12_7_notebook_cloning.png"/><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SVN12_7_notebook_cell_cultures.png"/><br />
<div style="clear:both"></div><br />
</td><br />
</tr><br />
<br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 1.</b> Schematic presentation of methods used for cloning and culturing eukaryotic cells.<br />
</td><br />
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<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/8/84/SVN12_7_notebook_switch.png"/></td><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/e/e7/SVN12_7_notebook_safety_mech.png"/></td><br />
</tr><br />
<tr><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/9/91/SVN12_7_notebook_encapsulation.png"/></td><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/c/c8/SVN12_7_notebook_effectors.png"/></td><br />
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<table class="invisible" style="width:80%;"><br />
<tbody><br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 2.</b> Schematic presentation of methods used for characterizing the switch, safety mechanisms, microencapsulation and effectors.</td><br />
</tr><br />
</tbody><br />
</table><br />
<br />
<h2><a name="cloning"> Cloning </a></h2><br />
<h3>Plasmid DNA isolation</h3><br />
<p><b>MINI PREPs for analysis and sequencing</b></p><br />
<ol><br />
<li>A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). </li><br />
<li>Bacteria were grown over night at 37 °C with agitation. </li><br />
<li>Plasmid DNA was isolated from 6-10 mL of over-night culture with GeneJET plasmid miniprep kit according to the manufacturer's protocol. </li><br />
<li>Amounts ranging from 6-10 µg of plasmid DNA were obtained. </li><br />
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Fragment DNA isolation from agarose gel</h3><br />
<p><b>AGAROSE ELECTROPHORESIS</b></p><br />
<ol><br />
<li>A mixture of different sized DNA fragments was separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li><br />
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li><br />
</ol><br />
</br><br />
<p><b>FRAGMENT ISOLATION from agarose gel </b></p><br />
<ol><br />
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li><br />
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li><br />
<li>Purity and amount of DNA was determined using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Restriction digest</h3><br />
<ol><br />
<li>To digest the desired DNA restriction reactions were prepared as follows: </li><br />
<ul style="margin-left:15px;"> <b>for analysis of cloned DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>0.5 µL restriction enzyme</li><br />
<br />
<li>Bring volume to 20 µL with nuclease-free water.</li><br />
</ul><br />
<i>or</i><br />
<ul style="margin-left:15px;"> <b>for isolation of specific DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>up to 2 µL restriction enzyme </li><br />
<br />
<li>Bring volume to 50 µL with nuclease-free water.</li><br />
</ul><br />
</li><br />
<li>The sample was incubated at optimal temperature for the restriction enzymes.</li><br />
<li>Analysis of fragmented DNA was done by gel electrophoreses. </li><br />
<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<h3>PCR reaction</h3><br />
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p><br />
<ol><br />
<br />
<li>The master mix for reactions with Phusion DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (1-10 ng)</li><br />
<br />
<li>both primers (0,4 pmol/µl )</li><br />
<br />
<li>1x Phusion HF buffer</li><br />
<br />
<li>0,2 µM dNTPs</li><br />
<br />
<li>Phusion polymerase (0,02 U/ µl) and </li><br />
<br />
<li>MQ up to final volume of 25 µl</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with AccuPrime DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (10 ng),</li><br />
<br />
<li>both primers (0,4 pmol/µl ),</li><br />
<br />
<li>1xRnx mix,</li><br />
<br />
<li>enzyme (0,05 U/ µl) and</li><br />
<br />
<li>MQ up to final volume of 50 µl.</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with Taq DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>both primers (0,4 pmol/µl),</li><br />
<br />
<li>1x Taq PCR buffer II,</li><br />
<br />
<li>0,2 µM dNTPs,</li><br />
<br />
<li>5mM MgSO4,</li><br />
<br />
<li>enzyme (0,125 U/ µl) and</li><br />
<br />
<li>MQ up to total volume of 20 µl.</li><br />
<br />
<li>Then the bacterial colony was added to the reaction mix.</li> <br />
</ul><br />
</li><br />
<br />
<li>All temperature profiles were optimized according to manufacturer’s protocol,the melting temperature of primers, and the length of the desired PCR products. Reactions were performed in the Applied Biosystems Veriti 96 well thermal cycler. </li><br />
</ol><br />
</br><br />
<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p><br />
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p><br />
<h3>Gibson assembly </h3><br />
<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p><br />
<ol><br />
<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li><br />
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Ligation</h3><br />
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p><br />
<ol><br />
<li>Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA). </li><br />
<li>A ligation mixture was prepared: </li><br />
<br/><br />
1X ligase buffer (10X)<br />
<br/><br />
1 µL T4 ligase (3 U/µL)<br />
<br/><br />
Bring volume to 10 or 20 µL with nuclease-free water.<br />
</li></br><br />
<i>or</i><br />
<br />
<li>Blunt-end ligation reactions were incubated at 17 °C for 4 to 18 hours. </li><br />
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li><br />
</ol><br />
</br><br />
<br />
<h3> Culturing bacteria</h3><br />
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p><br />
<p><b> Growth media for bacteria</b></p><br />
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p><br />
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p><br />
</br><br />
<br />
<h3>Transformation of bacteria</h3><br />
<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p><br />
<ol><br />
<li>100 µL of competent cells were thawed on ice. </li><br />
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li><br />
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li><br />
<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li><br />
<li>Cooled for 3 minutes on ice. </li><br />
<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li><br />
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li><br />
</ol><br />
</br><br />
<br />
<h3>Glycerol stock for long term storage of bacteria</h3><br />
<ol><br />
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li><br />
<li>Mixed and incubated at room temperature for 30 minutes. </li><br />
<li>Afterwards the glycerol stock was stored at -80 °C. </li><br />
</ol><br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="cellcultures"> Cell cultures </a></h2><br />
<h3>Eucaryotic cell lines and cultivation</h3><br />
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p><br />
</br><br />
<br />
<h3>Subculturing monolayer cell cultures</h3><br />
<ol><br />
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li><br />
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li><br />
<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li><br />
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li><br />
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li><br />
<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li><br />
<li>Remove the trypsin-containing medium from the centrifuge tube. </li><br />
<li>Resuspend the cell pellet in fresh medium. </li><br />
<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li><br />
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li><br />
</ol><br />
</br><br />
<h3>Cell plating</h3><br />
<br />
<ol><br />
<li>Count cells. </li><br />
<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li><br />
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li><br />
</ol><br />
<br/><br />
<p><b>Media and buffers</b><br />
<br/><br />
<br><b>DMEM</b> supplemented with: 1 % L-Glutamine (GlutaMax), 10 % FBS, Optionally: 1% Pen/Strep.<br />
<br><b>RPMI</b> supplemented with: 1 % L-Glutamine (GlutaMax), 20 % FBS.</p><br />
<br />
<br/><br />
<h3>Transfection</h3><br />
TABLE: Transfection mixtures for different culture format<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Culture format</th><br />
<th>jetPEI reagent per µg of DNA (µL)</th><br />
<th>Typical amount of DNA (ng)</th><br />
<th>Volume of 150 mM NaCl solution for DNA and jetPEI (µL)</th><br />
<th>Total transfection mixture volume (µL)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">96-well</td><br />
<td class="normal">2</td><br />
<td class="normal">200</td><br />
<td class="normal">10</td><br />
<td class="normal">20</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">24-well and 8-well microscope chamber</td><br />
<td class="normal">2</td><br />
<td class="normal">500</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal">12-well</td><br />
<td class="normal">2</td><br />
<td class="normal">1000</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">6-well</td><br />
<td class="normal">2</td><br />
<td class="normal">2000</td><br />
<td class="normal">100</td><br />
<td class="normal">200</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">10 cm</td><br />
<td class="normal">2</td><br />
<td class="normal">15000</td><br />
<td class="normal">250</td><br />
<td class="normal">500</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<ol><br />
<li>Dilute plasmid DNA to desired concentration in 150 mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Dilute jetPEI (PolyPlus) in 150mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Add the jetPEI solution to the DNA solution. </li><br />
<li>Vortex the solution immediately and spin down briefly. </li><br />
<li>Incubate for 15 to 30 minutes at room temperature. </li><br />
<li>Add the jetPEI/DNA mix to the cells in and gently swirl the plate. </li><br />
<li>Return the plate to a cell culture incubator. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="inductionsystems"> Induction systems </a></h2><br />
<br />
<h3>Induction systems</h3><br />
<br />
<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p><br />
<br />
<h3>Tetracycline, erythromycin and pristinamycin systems</h3><br />
<br />
<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Svn12_parts_inducibilni.png"></img><br />
<center><p><b> Figure 1: Induced expression of TAL</b>.</p></center><br />
<br />
<p>TABLE</p><br />
<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> System </th><br />
<br />
<th> Tetracycline </th><br />
<br />
<th> Erythromycin</th><br />
<br />
<th> Pristinamycin </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> Regulating protein </td><br />
<td class="normal"> TetR:KRAB </td><br />
<td class="normal"> E:KRAB </td><br />
<td class="normal"> PIP:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> TRE </td><br />
<td class="normal"> ETR </td><br />
<td class="normal"> PIR </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> Inducer </td><br />
<td class="normal"> Tetracycline or doxycycline </td><br />
<td class="normal"> Erythromycin </td><br />
<td class="normal"> Pristinamycin </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> pCMV_TRE_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_ETR_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> pCMV_TRE_TAL-B:VP16</td><br />
<td class="normal"> pCMV_ETR_TAL-B:VP16</td><br />
<td class="normal"> pCMV_PIR_TAL-A:VP16</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-B:KRAB </td><br />
<td class="normal"> pSV40_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-A:VP16 </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pSV40_ETR_TAL-B:KRAB </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<h3>Rapamycin system</h3><br />
<br />
<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p><br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> Regulating vector </th><br />
<br />
<th> HetAct </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> ZFHD </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> ZFHD_pMIN_TAL-A:KRAB </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> ZFHD_pMIN_TAL-B:VP16</td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
</br><br />
<h3>Induction of cells</h3><br />
<ol><br />
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li><br />
<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li><br />
</ol><br />
</br><br />
<br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Inductor</th><br />
<th>Stock solution (solvent)</th><br />
<th>Dilution (solvent)</th><br />
<th>Concentration in cell medium (% solvent)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">Rapalogue (AP21967)</td><br />
<td class="normal">1 mM (100% ethanol)</td><br />
<td class="normal">10 µM (1% ethanol)</td><br />
<td class="normal">1 µM (0,1% ethanol)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Doxycyclin</td><br />
<td class="normal">1 g/L (MQ)</td><br />
<td class="normal">10 mg/L (MQ)</td><br />
<td class="normal">1 mg/L (MQ)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Pristinamycin</td><br />
<td class="normal">50 g/L (100% DMSO)</td><br />
<td class="normal">20 mg/L (1% DMSO)</td><br />
<td class="normal">2 mg/L (0,1% DMSO)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Erythromycin</td><br />
<td class="normal">50 g/L (100% ethanol)</td><br />
<td class="normal">20 mg/L (1% ethanol)</td><br />
<td class="normal">2 mg/L (0,1% ethanol)</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<!-- ----------------------------------------------------------------- --><br />
</br><br/><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="effectors"> Effectors </a></h2><br />
<h3>Biological assay-anakinra </h3><br />
<ol><br />
<li>HEK293T cells, seeded in 6-well plate, were transfected with anakinra downstream of constitutive promoter. </li><br />
<li>Transfected cells were incubated for 48 h. </li><br />
<li>To detect anakinra's effect on NF-κB signalling pathway, other HEK293T cells were transfected with plasmid coding for Renilla luciferase and plasmid reporter with NF-κB-inducible firefly luciferase expression. HEK293T cells express IL-1R, so additional transfection with a receptor gene was notneeded. </li><br />
<li>After 24 h, the medium was removed from cells transfected with reporter plasmids and 90 μL of anakinra-producing cells' supernatant was added to these wells. </li><br />
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li><br />
</ol><br />
</br><br />
<br />
<h3> Biological assay-IFN-alpha </h3><br />
<ol><br />
<li>HEK293T cells transfected with eithera plasmid encoding IFN-alpha under the control of a constitutive promoter or an empty vector, and HEK293T cells transfected with the reporter vector were co-cultivated . </li><br />
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li><br />
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li><br />
<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li><br />
</ol><br />
</br><br />
<br />
<h3>ELISA for IFN-alpha</h3><br />
<ol><br />
<li>HEK293T cells where plated on 6 well plates and transfected with a plasmid coding for human IFN-alpha under the control of a constitutive promoter or a control plasmid (pcDNA3). </li><br />
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-fluorescence(The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in black 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega ) per well. </li><br />
<li>Fluorescence was measured using an automated plate reader. </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-luminescence (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two to three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li><br />
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Plate reader-absorbance (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li><br />
<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li><br />
<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C the absorbance was measured at 630 nm. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="microscopy"> Microscopy </a></h2><br />
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p><br />
</br><br />
<br />
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3><br />
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 200 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li><br />
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 nm and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell growth (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng GFP (pPCMV-GFP) (for transfection control). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a cell cultures were imaged. </li><br />
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite GFP reporter protein. Fluorescence emission was detected at 520-560 nm for GFP. Bright field images were used to visualize the number of cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell count (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates and transfected with different amounts of mGMK:TK30 (pPCMV_mGMK:TK30) as indicated in Figure legend. </li><br />
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the Figure legend. </li><br />
<li>After incubation the cells were resuspended by pipetting. </li><br />
<li>Cells suspensions were then mixed with trypan blue. </li><br />
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-detection of reporter proteins (The Switch) </h3><br />
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p><br />
<ol><br />
<li>HEK293T cells were seeded in an 8-well microscope chamber or 12-well plate and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>Images of cells expressing reporters were taken two days after transfection and then each day for 5 days. </li><br />
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-alginate degradation (Microencapsulation) </h3><br />
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation upon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p><br />
<ol><br />
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into well. </li><br />
<li>After the dye was evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added. </li><br />
<li>The microscope was set to capture images every 20 seconds. </li><br />
<li>Screenshots were collected for at least 15 minutes. </li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Microscopy-secreted alginate lyase enzymatic activity (Microencapsulation)</h3><br />
<ol><br />
<li>HEK293T cells were seeded 1×10<sup>6</sup> on 10 cm cell culture dish and grown in DMEM medium supplemented with 10 % FBS.</li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection).</li><br />
<li>Protein production lasted for 72 hours.</li><br />
<li>Cell supernatants were collected and concentrated 50-times using Sartorius Vivaspin 6 concentrators.</li><br />
<li>Alginate beads were produced with Büchi BIOTECH Encapsulator (see Microencapsulation: Encapsulation procedure 1.-6.).</li><br />
<li>Beads were incubated with concentrated supernatants for 72 hours in an 8-well microscope chamber.</li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into wells.</li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC detection. Fluorescence emission was detected at 520-560 nm.</li><br />
<li>Beads' diameters were assessed using Leica LAS Image Analysis software.</li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3><br />
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both live and dead cells, while 7-AAD stains dead cells only.</p><br />
<ol><br />
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS. </li><br />
<li>200 µL of the microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber. </li><br />
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added to one well. </li><br />
<li>Encapsulated cells were protected from direct light and stained for 30 minutes at 37 °C. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 543-nm line of HeNe laser was used to excite 7-AAD. </li><br />
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="flowcytometry"> Flow cytometry </a></h2><br />
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p><br />
</br><br />
<br />
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labeled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with phosphatidylserine exposed on their surface.</p><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates. </li><br />
<li>Cells were transfected with pCMV-mGMK_TK30 and treated with ganciclovir. Concentrations of ganciclovir and plasmids are indicated in Figure legends. </li><br />
<li>After incubation the cells were washed with PBS buffer and resuspended by pipetting. </li><br />
<li>Cells were pelleted with centrifugation at 1200 rpm. </li><br />
<li>The cell pellet was washed in 1x Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). </li><br />
<li>The pellet was then resuspended in 1x Annexin Binding Buffer and PE-Annexin V (5 µl per 100 μl cell suspension) was added. </li><br />
<li>Samples were incubated for 20 minutes in the dark at room temperature and then immediately analyzed with a flow cytometer. </li><br />
<li>Along with site and forward scatter, the signal in the FL2 channel (540-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p><br />
<ol><br />
<li>HEK293T cells seeded in 12-well plates were transfected with plasmids expressing MICA (pPCMV-MICA_pcDNA3).</li><br />
<li>Two days after transfection with CMV-MICA_pcDNA3, cells were resuspended in PBS at final concentration 1x10<sup>6</sup> cells/mL, stained with CFSE (0,6μM) and incubated for 10 minutes at 37°C. Staining was quenched by the addition of 5 volumes of ice-cold culture media (RPMI+ 20% FBS) to the cells. After 5 minutes incubation on ice, cells were pelleted by centrifugation and then washed by resuspending the pellet in fresh media (RPMI+ 20% FBS) a further two times for a total of three washes. CFSE was used to discriminate between HEK293T and NK-92 cells or between NK target cells K562, which were used as a positive control, and NK-92 cells.</li><br />
<li>HEK293T cells or K562 cells were mixed with NK-92 cells in different ratios (1:1, 1:5, 1:10) and incubated for 4 hours at 37 °C in culture medium consisting of RPMI, 20% FBS and hIL-2 (100 U/ml).</li><br />
<li>After incubation of HEK293T or K562 cells with NK-92 cells, cells were treated with propidium iodide.</li><br />
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded.</li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3><br />
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p><br />
<ol><br />
<li>HEK293T cells were seeded in a 12 or 24-well plate and transfected with plasmids encoding the switch. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. </li><br />
<li>Medium with inducer or without the inducer was changed after two days of cultivation. </li><br />
<li>Cells were collected at different time points (2 days after induction and then 3 days after the second media change). </li><br />
<li>Cells were washed and resuspended in PBS buffer. </li><br />
<li>A 405 nm diode laser was used to excite tagBFP and a 488-nm diode laser was used for mCitirne. </li><br />
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="microencapsulation"> Microencapsulation </a></h2><br />
<h3>Cell preparation for encapsulation</h3><br />
<ol><br />
<li>HEK 293T cells were seeded 5x10⁵ per 10 cm cell culture dish (3 per encapsulation) and grown in DMEM medium supplemented with 10 % FBS. </li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection). </li><br />
<li>The medium was removed the next day.. Transfected cells were detached using 3 mL of trypsin solution and centrifuged after the addition of fresh medium to inactivate the trypsin. </li><br />
<li>Supernatant was removed and cells were resuspended in 15 mL DMEM with 10% FBS. </li><br />
<li>Cells were counted using Countess automated cell counter (Invitrogen). </li><br />
<li>HEK 293T cells were again centrifuged and supernatant was removed. </li><br />
<li>Cells were resuspended in 2 mL of pre-warmed MOPS buffer. </li><br />
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li><br />
</ol><br />
</br><br />
<br />
<h3>Encapsulation</h3><br />
<ol><br />
<li>The encapsulator was equipped with a 200-µm nozzle. </li><br />
<li>The reactor vessel was filled with 225 mL 100 mM CaCl2. </li><br />
<li>Cell-alginate mixture was transferred to a 20 mL syringe with a Luer lock. </li><br />
<li>The syringe was connected to the bead producing unit (BPU). </li><br />
<li>Microcapsules were produced at a flow rate of 12-14 units, vibration frequency 1030-1100 Hz and voltage for bead dispersion 900-1300 V. </li><br />
<li>Polymerization lasted for 10 minutes. </li><br />
<li>The polymerization solution was drained and 75 mL of 0,05% poly-L-lysine (PLL) solution was added. </li><br />
<li>Beads were incubated in PLL solution for 10 minutes. </li><br />
<li>The PLL solution was removed and beads were washed twice (for 1 and for 5 minutes) with 150 mL of MOPS buffer. </li><br />
<li>100 mL of 0,03% alginate was added and beads were incubated for 10 minutes. </li><br />
<li>Alginate solution was drained and beads were washed once with 150 mL of MOPS buffer for 1 minute. </li><br />
<li>150 mL of depolymerization solution was added for 10 minutes. </li><br />
<li>Depolymerization solution was removed and capsules were resuspended in 150 mL MOPS and collected in a bead collection flask. </li><br />
<li>MOPS was removed and microcapsules were transferred to T-75 with 10 mL DMEM, 10% FBS media supplemented with penicillin and streptomycin. </li><br />
</ol><br />
<br />
<br/><br />
<p><b>Buffers and solutions</b><br />
<br/><br />
<br><b>10 mM MOPS buffer</b> (pH = 7,2)<br />
<br><b>MOPS buffer with NaCl</b> (pH = 7,2): 10 mM MOPS, 0,85% NaCl<br />
<br><b>Polymerisation solution</b> (pH = 7,2): 10 mM MOPS, 100 mM CaCl2<br />
<br><b>Depolymerisation solution</b> (pH = 7,2): 10 mM MOPS, 50 mM Na3-citrate, 0,45% NaCl<br />
<br><b>0,05% poly-L-lysine</b> (15-30 kDa) in MOPS buffer (pH = 7,3) <br />
<br><b>0,03% alginate</b> in MOPS buffer (pH = 7,2)<br />
<br><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2)</p><br />
</br><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="proteindetection"> Protein detection </a></h2><br />
<h3>SDS-PAGE</h3><br />
<ol><br />
<li>Samples were loaded on a 12% acrylamide gel and ran at a constant voltage (200 V) for 1 h. </li><br />
<li>Proteins were then blotted on a nitrocellulose membrane at a constant current (350 mA) for 1 h. </li><br />
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li><br />
</ol><br />
</br><br />
<br />
<h3>Immunodetection </h3><br />
<ol><br />
<li>All proteins to be detected had a Myc tag at the C-terminus. </li><br />
<li>The membrane was incubated with primary antibodies (rabbit anti-Myc diluted 1:500) overnight at 4 °C and 150 rpm. Membrane was washed. </li><br />
<li>Washing in wash buffer three times for 5 minutes each.</li><br />
<li>Membrane was incubated with secondary antibodies (anti-rabbit secondary antibodies, conjugated with HRP, diluted 1:3000) for 45 minutes at room temperature and 150 rpm. </li><br />
<li>HRP activity was detected by addition of SuperSignal West Femto or Pico Substrate (Thermo Scientific). Images were captured with Syngene G:Box chemiluminescent imaging system. </li><br />
</ol><br />
<p><b>Buffers and solutions </b></p><br />
<p><b>Wash buffer</b>: 1x PBS, 0,01% (v/v) Tween 20</p><br />
</br><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Clackson, T. (2000) Regulated gene expression systems. <i> Gene ther.</i> <b>7</b>, 120–125.<br/><br/><br />
Deuschle, U., Meyer, W.K. and Thiesen, R. (1995) Tetracycline-reversible silencing of eukaryotic promoters. <i>Mol. Cell. Biol. </i> <b>15</b>, 1907–1914.<br/><br/><br />
Kramer, B.P., Fischer, C. and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells. <i> Biotech. Bioeng.</i> <b>87</b>, 478–484.<br/><br/><br />
Pollock, R. and Clackson, T. (2002) Dimerizer-regulated gene expression. <i> Curr. Opin. Biotech. </i> <b>13</b>, 459–467.<br/><br/><br />
Gibson, D.G., Young, L., Chuang, R., Venter J.C., Hutchison III, C. A. and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature methods.</i> <b>6</b>, 343–345.<br />
</p><br />
<br />
<br />
<br/><br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'>Lablog >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/Notebook
Team:Slovenia/Notebook
2012-10-26T16:59:00Z
<p>Dusanv: </p>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br />
<br />
<br/><br />
<h1><a name="naslov"></a>Experimental methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#cloning">Cloning</a><br/></li><br />
<li><a href="#cellcultures">Cell cultures</a></li><br />
<li><a href="#inductionsystems">Induction systems</a></li><br />
<li><a href="#effectors">Effectors</a></li><br />
<li><a href="#microscopy">Microscopy</a></li><br />
<li><a href="#flowcytometry">Flow cytometry</a></li><br />
<li><a href="#microencapsulation">Microencapsulation</a></li><br />
<li><a href="#proteindetection">Protein detection</a></li><br />
</ul><br />
</p><br />
<br />
<br />
<br />
<br />
<!-- figure 1 --><br />
<p><br />
<table class="invisible" style="width:80%;"><br />
<tbody class="invisible"><br />
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<td class="invisible"><br />
<img style="float:left;" src="https://static.igem.org/mediawiki/2012/a/a7/SVN12_7_notebook_cloning.png"/><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SVN12_7_notebook_cell_cultures.png"/><br />
<div style="clear:both"></div><br />
</td><br />
</tr><br />
<br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 1.</b> Schematic presentation of methods used for cloning and culturing eukaryotic cells.<br />
</td><br />
</tr><br />
</tbody><br />
</table> <br />
</p><br />
<!-- end figure 1--><br />
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<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/8/84/SVN12_7_notebook_switch.png"/></td><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/e/e7/SVN12_7_notebook_safety_mech.png"/></td><br />
</tr><br />
<tr><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/9/91/SVN12_7_notebook_encapsulation.png"/></td><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/c/c8/SVN12_7_notebook_effectors.png"/></td><br />
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<br />
<br />
<table class="invisible" style="width:80%;"><br />
<tbody><br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 2.</b> Schematic presentation of methods used for characterizing the switch, safety mechanisms, microencapsulation and effectors.</td><br />
</tr><br />
</tbody><br />
</table><br />
<br />
<h2><a name="cloning"> Cloning </a></h2><br />
<h3>Plasmid DNA isolation</h3><br />
<p><b>MINI PREPs for analysis and sequencing</b></p><br />
<ol><br />
<li>A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). </li><br />
<li>Bacteria were grown over night at 37 °C with agitation. </li><br />
<li>Plasmid DNA was isolated from 6-10 mL of over-night culture with GeneJET plasmid miniprep kit according to the manufacturer's protocol. </li><br />
<li>Amounts ranging from 6-10 µg of plasmid DNA were obtained. </li><br />
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Fragment DNA isolation from agarose gel</h3><br />
<p><b>AGAROSE ELECTROPHORESIS</b></p><br />
<ol><br />
<li>A mixture of different sized DNA fragments was separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li><br />
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li><br />
</ol><br />
</br><br />
<p><b>FRAGMENT ISOLATION from agarose gel </b></p><br />
<ol><br />
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li><br />
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li><br />
<li>Purity and amount of DNA was determined using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Restriction digest</h3><br />
<ol><br />
<li>To digest the desired DNA restriction reactions were prepared as follows: </li><br />
<ul style="margin-left:15px;"> <b>for analysis of cloned DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>0.5 µL restriction enzyme</li><br />
<br />
<li>Bring volume to 20 µL with nuclease-free water.</li><br />
</ul><br />
<i>or</i><br />
<ul style="margin-left:15px;"> <b>for isolation of specific DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>up to 2 µL restriction enzyme </li><br />
<br />
<li>Bring volume to 50 µL with nuclease-free water.</li><br />
</ul><br />
</li><br />
<li>The sample was incubated at optimal temperature for the restriction enzymes.</li><br />
<li>Analysis of fragmented DNA was done by gel electrophoreses. </li><br />
<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<h3>PCR reaction</h3><br />
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p><br />
<ol><br />
<br />
<li>The master mix for reactions with Phusion DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (1-10 ng)</li><br />
<br />
<li>both primers (0,4 pmol/µl )</li><br />
<br />
<li>1x Phusion HF buffer</li><br />
<br />
<li>0,2 µM dNTPs</li><br />
<br />
<li>Phusion polymerase (0,02 U/ µl) and </li><br />
<br />
<li>MQ up to final volume of 25 µl</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with AccuPrime DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (10 ng),</li><br />
<br />
<li>both primers (0,4 pmol/µl ),</li><br />
<br />
<li>1xRnx mix,</li><br />
<br />
<li>enzyme (0,05 U/ µl) and</li><br />
<br />
<li>MQ up to final volume of 50 µl.</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with Taq DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>both primers (0,4 pmol/µl),</li><br />
<br />
<li>1x Taq PCR buffer II,</li><br />
<br />
<li>0,2 µM dNTPs,</li><br />
<br />
<li>5mM MgSO4,</li><br />
<br />
<li>enzyme (0,125 U/ µl) and</li><br />
<br />
<li>MQ up to total volume of 20 µl.</li><br />
<br />
<li>Then the bacterial colony was added to the reaction mix.</li> <br />
</ul><br />
</li><br />
<br />
<li>All temperature profiles were optimized according to manufacturer’s protocol,the melting temperature of primers, and the length of the desired PCR products. Reactions were performed in the Applied Biosystems Veriti 96 well thermal cycler. </li><br />
</ol><br />
</br><br />
<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p><br />
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p><br />
<h3>Gibson assembly </h3><br />
<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p><br />
<ol><br />
<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li><br />
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Ligation</h3><br />
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p><br />
<ol><br />
<li>Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA). </li><br />
<li>A ligation mixture was prepared: </li><br />
<br/><br />
1X ligase buffer (10X)<br />
<br/><br />
1 µL T4 ligase (3 U/µL)<br />
<br/><br />
Bring volume to 10 or 20 µL with nuclease-free water.<br />
</li></br><br />
<i>or</i><br />
<br />
<li>Blunt-end ligation reactions were incubated at 17 °C for 4 to 18 hours. </li><br />
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li><br />
</ol><br />
</br><br />
<br />
<h3> Culturing bacteria</h3><br />
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p><br />
<p><b> Growth media for bacteria</b></p><br />
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p><br />
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p><br />
</br><br />
<br />
<h3>Transformation of bacteria</h3><br />
<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p><br />
<ol><br />
<li>100 µL of competent cells were thawed on ice. </li><br />
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li><br />
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li><br />
<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li><br />
<li>Cooled for 3 minutes on ice. </li><br />
<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li><br />
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li><br />
</ol><br />
</br><br />
<br />
<h3>Glycerol stock for long term storage of bacteria</h3><br />
<ol><br />
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li><br />
<li>Mixed and incubated at room temperature for 30 minutes. </li><br />
<li>Afterwards the glycerol stock was stored at -80 °C. </li><br />
</ol><br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="cellcultures"> Cell cultures </a></h2><br />
<h3>Eucaryotic cell lines and cultivation</h3><br />
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p><br />
</br><br />
<br />
<h3>Subculturing monolayer cell cultures</h3><br />
<ol><br />
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li><br />
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li><br />
<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li><br />
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li><br />
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li><br />
<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li><br />
<li>Remove the trypsin-containing medium from the centrifuge tube. </li><br />
<li>Resuspend the cell pellet in fresh medium. </li><br />
<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li><br />
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li><br />
</ol><br />
</br><br />
<h3>Cell plating</h3><br />
<br />
<ol><br />
<li>Count cells. </li><br />
<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li><br />
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li><br />
</ol><br />
<br/><br />
<p><b>Media and buffers</b><br />
<br/><br />
<br><b>DMEM</b> supplemented with: 1 % L-Glutamine (GlutaMax), 10 % FBS, Optionally: 1% Pen/Strep.<br />
<br><b>RPMI</b> supplemented with: 1 % L-Glutamine (GlutaMax), 20 % FBS.</p><br />
<br />
<br/><br />
<h3>Transfection</h3><br />
TABLE: Transfection mixtures for different culture format<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Culture format</th><br />
<th>jetPEI reagent per µg of DNA (µL)</th><br />
<th>Typical amount of DNA (ng)</th><br />
<th>Volume of 150 mM NaCl solution for DNA and jetPEI (µL)</th><br />
<th>Total transfection mixture volume (µL)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">96-well</td><br />
<td class="normal">2</td><br />
<td class="normal">200</td><br />
<td class="normal">10</td><br />
<td class="normal">20</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">24-well and 8-well microscope chamber</td><br />
<td class="normal">2</td><br />
<td class="normal">500</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal">12-well</td><br />
<td class="normal">2</td><br />
<td class="normal">1000</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">6-well</td><br />
<td class="normal">2</td><br />
<td class="normal">2000</td><br />
<td class="normal">100</td><br />
<td class="normal">200</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">10 cm</td><br />
<td class="normal">2</td><br />
<td class="normal">15000</td><br />
<td class="normal">250</td><br />
<td class="normal">500</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<ol><br />
<li>Dilute plasmid DNA to desired concentration in 150 mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Dilute jetPEI (PolyPlus) in 150mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Add the jetPEI solution to the DNA solution. </li><br />
<li>Vortex the solution immediately and spin down briefly. </li><br />
<li>Incubate for 15 to 30 minutes at room temperature. </li><br />
<li>Add the jetPEI/DNA mix to the cells in and gently swirl the plate. </li><br />
<li>Return the plate to a cell culture incubator. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="inductionsystems"> Induction systems </a></h2><br />
<br />
<h3>Induction systems</h3><br />
<br />
<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p><br />
<br />
<h3>Tetracycline, erythromycin and pristinamycin systems</h3><br />
<br />
<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Svn12_parts_inducibilni.png"></img><br />
<center><p><b> Figure 1: Induced expression of TAL</b>.</p></center><br />
<br />
<p>TABLE</p><br />
<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> System </th><br />
<br />
<th> Tetracycline </th><br />
<br />
<th> Erythromycin</th><br />
<br />
<th> Pristinamycin </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> Regulating protein </td><br />
<td class="normal"> TetR:KRAB </td><br />
<td class="normal"> E:KRAB </td><br />
<td class="normal"> PIP:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> TRE </td><br />
<td class="normal"> ETR </td><br />
<td class="normal"> PIR </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> Inducer </td><br />
<td class="normal"> Tetracycline or doxycycline </td><br />
<td class="normal"> Erythromycin </td><br />
<td class="normal"> Pristinamycin </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> pCMV_TRE_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_ETR_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> pCMV_TRE_TAL-B:VP16</td><br />
<td class="normal"> pCMV_ETR_TAL-B:VP16</td><br />
<td class="normal"> pCMV_PIR_TAL-A:VP16</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-B:KRAB </td><br />
<td class="normal"> pSV40_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-A:VP16 </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pSV40_ETR_TAL-B:KRAB </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<h3>Rapamycin system</h3><br />
<br />
<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p><br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> Regulating vector </th><br />
<br />
<th> HetAct </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> ZFHD </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> ZFHD_pMIN_TAL-A:KRAB </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> ZFHD_pMIN_TAL-B:VP16</td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
</br><br />
<h3>Induction of cells</h3><br />
<ol><br />
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li><br />
<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li><br />
</ol><br />
</br><br />
<br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Inductor</th><br />
<th>Stock solution (solvent)</th><br />
<th>Dilution (solvent)</th><br />
<th>Concentration in cell medium (% solvent)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">Rapalogue (AP21967)</td><br />
<td class="normal">1 mM (100% ethanol)</td><br />
<td class="normal">10 µM (1% ethanol)</td><br />
<td class="normal">1 µM (0,1% ethanol)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Doxycyclin</td><br />
<td class="normal">1 g/L (MQ)</td><br />
<td class="normal">10 mg/L (MQ)</td><br />
<td class="normal">1 mg/L (MQ)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Pristinamycin</td><br />
<td class="normal">50 g/L (100% DMSO)</td><br />
<td class="normal">20 mg/L (1% DMSO)</td><br />
<td class="normal">2 mg/L (0,1% DMSO)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Erythromycin</td><br />
<td class="normal">50 g/L (100% ethanol)</td><br />
<td class="normal">20 mg/L (1% ethanol)</td><br />
<td class="normal">2 mg/L (0,1% ethanol)</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<!-- ----------------------------------------------------------------- --><br />
</br><br/><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="effectors"> Effectors </a></h2><br />
<h3>Biological assay-anakinra </h3><br />
<ol><br />
<li>HEK293T cells, seeded in 6-well plate, were transfected with anakinra downstream of constitutive promoter. </li><br />
<li>Transfected cells were incubated for 48 h. </li><br />
<li>To detect anakinra's effect on NF-κB signalling pathway, other HEK293T cells were transfected with plasmid coding for Renilla luciferase and plasmid reporter with NF-κB-inducible firefly luciferase expression. HEK293T cells express IL-1R, so additional transfection with a receptor gene was notneeded. </li><br />
<li>After 24 h, the medium was removed from cells transfected with reporter plasmids and 90 μL of anakinra-producing cells' supernatant was added to these wells. </li><br />
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li><br />
</ol><br />
</br><br />
<br />
<h3> Biological assay-IFN-alpha </h3><br />
<ol><br />
<li>HEK293T cells transfected with eithera plasmid encoding IFN-alpha under the control of a constitutive promoter or an empty vector, and HEK293T cells transfected with the reporter vector were co-cultivated . </li><br />
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li><br />
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li><br />
<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li><br />
</ol><br />
</br><br />
<br />
<h3>ELISA for IFN-alpha</h3><br />
<ol><br />
<li>HEK293T cells where plated on 6 well plates and transfected with a plasmid coding for human IFN-alpha under the control of a constitutive promoter or a control plasmid (pcDNA3). </li><br />
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-fluorescence(The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in black 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega ) per well. </li><br />
<li>Fluorescence was measured using an automated plate reader. </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-luminescence (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two to three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li><br />
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Plate reader-absorbance (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li><br />
<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li><br />
<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C the absorbance was measured at 630 nm. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="microscopy"> Microscopy </a></h2><br />
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p><br />
</br><br />
<br />
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3><br />
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 200 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li><br />
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 nm and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell growth (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng GFP (pPCMV-GFP) (for transfection control). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a cell cultures were imaged. </li><br />
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite GFP reporter protein. Fluorescence emission was detected at 520-560 nm for GFP. Bright field images were used to visualize the number of cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell count (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates and transfected with different amounts of mGMK:TK30 (pPCMV_mGMK:TK30) as indicated in Figure legend. </li><br />
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the Figure legend. </li><br />
<li>After incubation the cells were resuspended by pipetting. </li><br />
<li>Cells suspensions were then mixed with trypan blue. </li><br />
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-detection of reporter proteins (The Switch) </h3><br />
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p><br />
<ol><br />
<li>HEK293T cells were seeded in an 8-well microscope chamber or 12-well plate and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>Images of cells expressing reporters were taken two days after transfection and then each day for 5 days. </li><br />
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-alginate degradation (Microencapsulation) </h3><br />
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation upon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p><br />
<ol><br />
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into well. </li><br />
<li>After the dye was evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added. </li><br />
<li>The microscope was set to capture images every 20 seconds. </li><br />
<li>Screenshots were collected for at least 15 minutes. </li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Microscopy-secreted alginate lyase enzymatic activity (Microencapsulation)</h3><br />
<ol><br />
<li>HEK293T cells were seeded 1×10<sup>6</sup> on 10 cm cell culture dish and grown in DMEM medium supplemented with 10 % FBS.</li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection).</li><br />
<li>Protein production lasted for 72 hours.</li><br />
<li>Cell supernatants were collected and concentrated 50-times using Sartorius Vivaspin 6 concentrators.</li><br />
<li>Alginate beads were produced with Büchi BIOTECH Encapsulator (see Microencapsulation: Encapsulation procedure 1.-6.).</li><br />
<li>Beads were incubated with concentrated supernatants for 72 hours in an 8-well microscope chamber.</li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into wells.</li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC detection. Fluorescence emission was detected at 520-560 nm.</li><br />
<li>Beads' diameters were assessed using Leica LAS Image Analysis software.</li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3><br />
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both living and dead cells, while 7-AAD stains dead cells only.</p><br />
<ol><br />
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS. </li><br />
<li>200 µL of the microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber. </li><br />
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added to one well. </li><br />
<li>Encapsulated cells were protected from direct light and stained for 30 min at 37 °C. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 543-nm line of HeNe laser was used to excite 7-AAD. </li><br />
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li><br />
</ol><br />
</br><br />
<br />
<br />
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<br />
<h2><a name="flowcytometry"> Flow cytometry </a></h2><br />
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p><br />
</br><br />
<br />
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labelled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with phosphatidylserine exposed on their surface.</p><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates. </li><br />
<li>Cells were transfected with pCMV-mGMK_TK30 and treated with ganciclovir. Concentrations of ganciclovir and plasmids are indicated in figure legends. </li><br />
<li>After incubation the cells were washed with PBS buffer and resuspended by pipetting. </li><br />
<li>Cells were pelleted with centrifugation at 1200 rpm. </li><br />
<li>The cell pellet was washed in 1x Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). </li><br />
<li>The pellet was then resuspended in 1x Annexin Binding Buffer and PE-Annexin V (5 µl per 100 μl cell suspension) was added. </li><br />
<li>Samples were incubated for 20 minutes in the dark at room temperature and then immediately analyzed with a flow cytometer. </li><br />
<li>Along with site and forward scatter, the signal in the FL2 channel (540-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p><br />
<ol><br />
<li>HEK293T cells seeded in 12-well plates were transfected with plasmids expressing MICA (pPCMV-MICA_pcDNA3) and/or a blue fluorescent protein (BFP) (pPCMV-BFP). BFP was used to discriminate between HEK293T and NK-92 cells. </li><br />
<li>Two days after transfection cells were mixed with NK-92 cells in different ratios (1:1, 1:5, 1:10) and incubated for 4 hours at 37 °C in culture medium consisting of RPMI, 20% FBS and hIL-2 (100 U/ml). </li><br />
<li>After incubation of HEK293T with NK-92 cells, cells were treated with propidium iodide. </li><br />
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3><br />
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p><br />
<ol><br />
<li>HEK293T cells were seeded in a 12 or 24-well plate and transfected with plasmids encoding the switch. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. </li><br />
<li>Medium with inducer or without the inducer was changed after two days of cultivation. </li><br />
<li>Cells were collected at different time points (2 days after induction and then 3 days after the second media change). </li><br />
<li>Cells were washed and resuspended in PBS buffer. </li><br />
<li>A 405 nm diode laser was used to excite tagBFP and a 488-nm diode laser was used for mCitirne. </li><br />
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="microencapsulation"> Microencapsulation </a></h2><br />
<h3>Cell preparation for encapsulation</h3><br />
<ol><br />
<li>HEK 293T cells were seeded 5x10⁵ per 10 cm cell culture dish (3 per encapsulation) and grown in DMEM medium supplemented with 10 % FBS. </li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection). </li><br />
<li>The medium was removed the next day.. Transfected cells were detached using 3 mL of trypsin solution and centrifuged after the addition of fresh medium to inactivate the trypsin. </li><br />
<li>Supernatant was removed and cells were resuspended in 15 mL DMEM with 10% FBS. </li><br />
<li>Cells were counted using Countess automated cell counter (Invitrogen). </li><br />
<li>HEK 293T cells were again centrifuged and supernatant was removed. </li><br />
<li>Cells were resuspended in 2 mL of pre-warmed MOPS buffer. </li><br />
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li><br />
</ol><br />
</br><br />
<br />
<h3>Encapsulation</h3><br />
<ol><br />
<li>The encapsulator was equipped with a 200-µm nozzle. </li><br />
<li>The reactor vessel was filled with 225 mL 100 mM CaCl2. </li><br />
<li>Cell-alginate mixture was transferred to a 20 mL syringe with a Luer lock. </li><br />
<li>The syringe was connected to the bead producing unit (BPU). </li><br />
<li>Microcapsules were produced at a flow rate of 12-14 units, vibration frequency 1030-1100 Hz and voltage for bead dispersion 900-1300 V. </li><br />
<li>Polymerisation lasted for 10 min. </li><br />
<li>The polymerization solution was drained and 75 mL of 0,05% poly-L-lysine (PLL) solution was added. </li><br />
<li>Beads were incubated in PLL solution for 10 minutes. </li><br />
<li>The PLL solution was removed and beads were washed twice (for 1 and for 5 min) with 150 mL of MOPS buffer. </li><br />
<li>100 mL of 0,03% alginate was added and beads were incubated for 10 min. </li><br />
<li>Alginate solution was drained and beads were washed once with 150 mL of MOPS buffer for 1 min. </li><br />
<li>150 mL of depolymerization solution was added for 10 min. </li><br />
<li>Depolymerization solution was removed and capsules were resuspended in 150 mL MOPS and collected in a bead collection flask. </li><br />
<li>MOPS was removed and microcapsules were transferred to T-75 with 10 mL DMEM, 10% FBS media supplemented with penicillin and streptomycin. </li><br />
</ol><br />
<br />
<br/><br />
<p><b>Buffers and solutions</b><br />
<br/><br />
<br><b>10 mM MOPS buffer</b> (pH = 7,2)<br />
<br><b>MOPS buffer with NaCl</b> (pH = 7,2): 10 mM MOPS, 0,85% NaCl<br />
<br><b>Polymerisation solution</b> (pH = 7,2): 10 mM MOPS, 100 mM CaCl2<br />
<br><b>Depolymerisation solution</b> (pH = 7,2): 10 mM MOPS, 50 mM Na3-citrate, 0,45% NaCl<br />
<br><b>0,05% poly-L-lysine</b> (15-30 kDa) in MOPS buffer (pH = 7,3) <br />
<br><b>0,03% alginate</b> in MOPS buffer (pH = 7,2)<br />
<br><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2)</p><br />
</br><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="proteindetection"> Protein detection </a></h2><br />
<h3>SDS-PAGE</h3><br />
<ol><br />
<li>Samples were loaded on a 12% acrylamide gel and ran at a constant voltage (200 V) for 1 h. </li><br />
<li>Proteins were then blotted on a nitrocellulose membrane at a constant current (350 mA) for 1 h. </li><br />
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li><br />
</ol><br />
</br><br />
<br />
<h3>Immunodetection </h3><br />
<ol><br />
<li>All proteins to be detected had a Myc tag at the C-terminus. </li><br />
<li>The membrane was incubated with primary antibodies (rabbit anti-Myc diluted 1:500) overnight at 4 °C and 150 rpm. Membrane was washed. </li><br />
<li>Washing in wash buffer three times for 5 min each.</li><br />
<li>Membrane was incubated with secondary antibodies (anti-rabbit secondary antibodies, conjugated with HRP, diluted 1:3000) for 45 min at room temperature and 150 rpm. </li><br />
<li>HRP activity was detected by addition of SuperSignal West Femto or Pico Substrate (Thermo Scientific). Images were captured with Syngene G:Box chemiluminescent imaging system. </li><br />
</ol><br />
<p><b>Buffers and solutions </b></p><br />
<p><b>Wash buffer</b>: 1x PBS, 0,01% (v/v) Tween 20</p><br />
</br><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Clackson, T. (2000) Regulated gene expression systems. <i> Gene ther.</i> <b>7</b>, 120–125.<br/><br/><br />
Deuschle, U., Meyer, W.K. and Thiesen, R. (1995) Tetracycline-reversible silencing of eukaryotic promoters. <i>Mol. Cell. Biol. </i> <b>15</b>, 1907–1914.<br/><br/><br />
Kramer, B.P., Fischer, C. and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells. <i> Biotech. Bioeng.</i> <b>87</b>, 478–484.<br/><br/><br />
Pollock, R. and Clackson, T. (2002) Dimerizer-regulated gene expression. <i> Curr. Opin. Biotech. </i> <b>13</b>, 459–467.<br/><br/><br />
Gibson, D.G., Young, L., Chuang, R., Venter J.C., Hutchison III, C. A. and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature methods.</i> <b>6</b>, 343–345.<br />
</p><br />
<br />
<br />
<br/><br />
<hr><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/Notebook
Team:Slovenia/Notebook
2012-10-26T16:41:21Z
<p>Dusanv: </p>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br />
<br />
<br/><br />
<h1><a name="naslov"></a>Experimental methods</h1><br />
<p><br />
<ul style="margin-left:15px;"><br />
<li><a href="#cloning">Cloning</a><br/></li><br />
<li><a href="#cellcultures">Cell cultures</a></li><br />
<li><a href="#inductionsystems">Induction systems</a></li><br />
<li><a href="#effectors">Effectors</a></li><br />
<li><a href="#microscopy">Microscopy</a></li><br />
<li><a href="#flowcytometry">Flow cytometry</a></li><br />
<li><a href="#microencapsulation">Microencapsulation</a></li><br />
<li><a href="#proteindetection">Protein detection</a></li><br />
</ul><br />
</p><br />
<br />
<br />
<br />
<br />
<!-- figure 1 --><br />
<p><br />
<table class="invisible" style="width:80%;"><br />
<tbody class="invisible"><br />
<tr class="invisible"><br />
<td class="invisible"><br />
<img style="float:left;" src="https://static.igem.org/mediawiki/2012/a/a7/SVN12_7_notebook_cloning.png"/><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SVN12_7_notebook_cell_cultures.png"/><br />
<div style="clear:both"></div><br />
</td><br />
</tr><br />
<br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 1.</b> Schematic presentation of methods used for cloning and culturing eukaryotic cells.<br />
</td><br />
</tr><br />
</tbody><br />
</table> <br />
</p><br />
<!-- end figure 1--><br />
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<br />
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<table class="invisible" style="width:77%;"><br />
<tbody class="invisible"><br />
<tr class="invisible"><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/8/84/SVN12_7_notebook_switch.png"/></td><br />
<td style="width:50%" class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/e/e7/SVN12_7_notebook_safety_mech.png"/></td><br />
</tr><br />
<tr><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/9/91/SVN12_7_notebook_encapsulation.png"/></td><br />
<td class="invisible"><img style="width:100%" src="https://static.igem.org/mediawiki/2012/c/c8/SVN12_7_notebook_effectors.png"/></td><br />
</tr> <br />
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</tbody><br />
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<br />
<br />
<table class="invisible" style="width:80%;"><br />
<tbody><br />
<tr class="invisible"><br />
<td class="invisible"><b>Figure 2.</b> Schematic presentation of methods used for characterizing the switch, safety mechanisms, microencapsulation and effectors.</td><br />
</tr><br />
</tbody><br />
</table><br />
<br />
<h2><a name="cloning"> Cloning </a></h2><br />
<h3>Plasmid DNA isolation</h3><br />
<p><b>MINI PREPs for analysis and sequencing</b></p><br />
<ol><br />
<li>A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). </li><br />
<li>Bacteria were grown over night at 37 °C with agitation. </li><br />
<li>Plasmid DNA was isolated from 6-10 mL of over-night culture with GeneJET plasmid miniprep kit according to the manufacturer's protocol. </li><br />
<li>Amounts ranging from 6-10 µg of plasmid DNA were obtained. </li><br />
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Fragment DNA isolation from agarose gel</h3><br />
<p><b>AGAROSE ELECTROPHORESIS</b></p><br />
<ol><br />
<li>A mixture of different sized DNA fragments was separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li><br />
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li><br />
</ol><br />
</br><br />
<p><b>FRAGMENT ISOLATION from agarose gel </b></p><br />
<ol><br />
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li><br />
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li><br />
<li>Purity and amount of DNA was determined using NanoDrop. </li><br />
</ol><br />
</br><br />
<h3>Restriction digest</h3><br />
<ol><br />
<li>To digest the desired DNA restriction reactions were prepared as follows: </li><br />
<ul style="margin-left:15px;"> <b>for analysis of cloned DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>0.5 µL restriction enzyme</li><br />
<br />
<li>Bring volume to 20 µL with nuclease-free water.</li><br />
</ul><br />
<i>or</i><br />
<ul style="margin-left:15px;"> <b>for isolation of specific DNA</b><br />
<li>2µl of the appropriate restriction buffer (10X)</li><br />
<br />
<li>up to 2 µL restriction enzyme </li><br />
<br />
<li>Bring volume to 50 µL with nuclease-free water.</li><br />
</ul><br />
</li><br />
<li>The sample was incubated at optimal temperature for the restriction enzymes.</li><br />
<li>Analysis of fragmented DNA was done by gel electrophoreses. </li><br />
<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<h3>PCR reaction</h3><br />
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p><br />
<ol><br />
<br />
<li>The master mix for reactions with Phusion DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (1-10 ng)</li><br />
<br />
<li>both primers (0,4 pmol/µl )</li><br />
<br />
<li>1x Phusion HF buffer</li><br />
<br />
<li>0,2 µM dNTPs</li><br />
<br />
<li>Phusion polymerase (0,02 U/ µl) and </li><br />
<br />
<li>MQ up to final volume of 25 µl</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with AccuPrime DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>DNA (10 ng),</li><br />
<br />
<li>both primers (0,4 pmol/µl ),</li><br />
<br />
<li>1xRnx mix,</li><br />
<br />
<li>enzyme (0,05 U/ µl) and</li><br />
<br />
<li>MQ up to final volume of 50 µl.</li><br />
</ul><br />
</li><br />
<br />
<li><br />
The master mix for reactions with Taq DNA polymerase contained:<br />
<ul style="margin-left:15px;"><br />
<li>both primers (0,4 pmol/µl),</li><br />
<br />
<li>1x Taq PCR buffer II,</li><br />
<br />
<li>0,2 µM dNTPs,</li><br />
<br />
<li>5mM MgSO4,</li><br />
<br />
<li>enzyme (0,125 U/ µl) and</li><br />
<br />
<li>MQ up to total volume of 20 µl.</li><br />
<br />
<li>Then the bacterial colony was added to the reaction mix.</li> <br />
</ul><br />
</li><br />
<br />
<li>All temperature profiles were optimized according to manufacturer’s protocol,the melting temperature of primers, and the length of the desired PCR products. Reactions were performed in the Applied Biosystems Veriti 96 well thermal cycler. </li><br />
</ol><br />
</br><br />
<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p><br />
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p><br />
<h3>Gibson assembly </h3><br />
<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p><br />
<ol><br />
<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li><br />
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Ligation</h3><br />
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p><br />
<ol><br />
<li>Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA). </li><br />
<li>A ligation mixture was prepared: </li><br />
<br/><br />
1X ligase buffer (10X)<br />
<br/><br />
1 µL T4 ligase (3 U/µL)<br />
<br/><br />
Bring volume to 10 or 20 µL with nuclease-free water.<br />
</li></br><br />
<i>or</i><br />
<br />
<li>Blunt-end ligation reactions were incubated at 17 °C for 4 to 18 hours. </li><br />
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li><br />
</ol><br />
</br><br />
<br />
<h3> Culturing bacteria</h3><br />
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p><br />
<p><b> Growth media for bacteria</b></p><br />
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p><br />
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p><br />
</br><br />
<br />
<h3>Transformation of bacteria</h3><br />
<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p><br />
<ol><br />
<li>100 µL of competent cells were thawed on ice. </li><br />
<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li><br />
<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li><br />
<li>The mixture was heat-shocked for 3 minutes at 42 °C. </li><br />
<li>Cooled for 3 minutes on ice. </li><br />
<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li><br />
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li><br />
</ol><br />
</br><br />
<br />
<h3>Glycerol stock for long term storage of bacteria</h3><br />
<ol><br />
<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li><br />
<li>Mixed and incubated at room temperature for 30 minutes. </li><br />
<li>Afterwards the glycerol stock was stored at -80 °C. </li><br />
</ol><br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="cellcultures"> Cell cultures </a></h2><br />
<h3>Eucaryotic cell lines and cultivation</h3><br />
<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>HEK293T</b> cell line is derived from HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p><br />
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p><br />
</br><br />
<br />
<h3>Subculturing monolayer cell cultures</h3><br />
<ol><br />
<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li><br />
<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li><br />
<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 minutes. </li><br />
<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li><br />
<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li><br />
<li>Centrifuge the cell suspension for 5 minutes at 1200 rpm. </li><br />
<li>Remove the trypsin-containing medium from the centrifuge tube. </li><br />
<li>Resuspend the cell pellet in fresh medium. </li><br />
<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li><br />
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li><br />
</ol><br />
</br><br />
<h3>Cell plating</h3><br />
<br />
<ol><br />
<li>Count cells. </li><br />
<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li><br />
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li><br />
</ol><br />
<br/><br />
<p><b>Media and buffers</b><br />
<br/><br />
<br><b>DMEM</b> supplemented with: 1 % L-Glutamine (GlutaMax), 10 % FBS, Optionally: 1% Pen/Strep.<br />
<br><b>RPMI</b> supplemented with: 1 % L-Glutamine (GlutaMax), 20 % FBS.</p><br />
<br />
<br/><br />
<h3>Transfection</h3><br />
TABLE: Transfection mixtures for different culture format<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Culture format</th><br />
<th>jetPEI reagent per µg of DNA (µL)</th><br />
<th>Typical amount of DNA (ng)</th><br />
<th>Volume of 150 mM NaCl solution for DNA and jetPEI (µL)</th><br />
<th>Total transfection mixture volume (µL)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">96-well</td><br />
<td class="normal">2</td><br />
<td class="normal">200</td><br />
<td class="normal">10</td><br />
<td class="normal">20</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">24-well and 8-well microscope chamber</td><br />
<td class="normal">2</td><br />
<td class="normal">500</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal">12-well</td><br />
<td class="normal">2</td><br />
<td class="normal">1000</td><br />
<td class="normal">50</td><br />
<td class="normal">100</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">6-well</td><br />
<td class="normal">2</td><br />
<td class="normal">2000</td><br />
<td class="normal">100</td><br />
<td class="normal">200</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">10 cm</td><br />
<td class="normal">2</td><br />
<td class="normal">15000</td><br />
<td class="normal">250</td><br />
<td class="normal">500</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<ol><br />
<li>Dilute plasmid DNA to desired concentration in 150 mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Dilute jetPEI (PolyPlus) in 150mM NaCl, vortex gently and spin down briefly. </li><br />
<li>Add the jetPEI solution to the DNA solution. </li><br />
<li>Vortex the solution immediately and spin down briefly. </li><br />
<li>Incubate for 15 to 30 minutes at room temperature. </li><br />
<li>Add the jetPEI/DNA mix to the cells in and gently swirl the plate. </li><br />
<li>Return the plate to a cell culture incubator. </li><br />
</ol><br />
<br />
<br />
<br/><br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="inductionsystems"> Induction systems </a></h2><br />
<br />
<h3>Induction systems</h3><br />
<br />
<p>To control our switch we needed a way to affect it from the outside. For this purpose we chose several inducible transcription systems where the controlled gene is expressed when a small molecule inducer (such as tetracycline) is present and is not expressed when the inducer is absent. We chose specific systems which do not cross react and whose inducers are orally bioavailable and safe for human use (Clackson, 2000). We decided for the systems based on tetracycline, pristinamycin, erythromycin and rapamycin analogs, as inducers. We then adapted these systems by cloning TAL regulators under their control to make them compatible with our genetic circuits. </p><br />
<br />
<h3>Tetracycline, erythromycin and pristinamycin systems</h3><br />
<br />
<p>The tetracycline, erythromycin and pristinamycin system all function in a similar manner. They are composed of a DNA binding protein (such as TetR) fused to a KRAB domain which reversibly binds a specific DNA sequence (TRE for example) and silences transcription from nearby promoters. The addition of an inducer causes the DNA binding domain to dissociate from the DNA and allows transcription to start. (Deuschle et al., 1995; Kramer et al., 2004) </p><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Svn12_parts_inducibilni.png"></img><br />
<center><p><b> Figure 1: Induced expression of TAL</b>.</p></center><br />
<br />
<p>TABLE</p><br />
<br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> System </th><br />
<br />
<th> Tetracycline </th><br />
<br />
<th> Erythromycin</th><br />
<br />
<th> Pristinamycin </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> Regulating protein </td><br />
<td class="normal"> TetR:KRAB </td><br />
<td class="normal"> E:KRAB </td><br />
<td class="normal"> PIP:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> TRE </td><br />
<td class="normal"> ETR </td><br />
<td class="normal"> PIR </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> Inducer </td><br />
<td class="normal"> Tetracycline or doxycycline </td><br />
<td class="normal"> Erythromycin </td><br />
<td class="normal"> Pristinamycin </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> pCMV_TRE_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_ETR_TAL-A:KRAB </td><br />
<td class="normal"> pCMV_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> pCMV_TRE_TAL-B:VP16</td><br />
<td class="normal"> pCMV_ETR_TAL-B:VP16</td><br />
<td class="normal"> pCMV_PIR_TAL-A:VP16</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-B:KRAB </td><br />
<td class="normal"> pSV40_PIR_TAL-B:KRAB </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pCMV_ETR_TAL-A:VP16 </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> </td><br />
<td class="normal"> pSV40_ETR_TAL-B:KRAB </td><br />
<td class="normal"> </td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<br />
<h3>Rapamycin system</h3><br />
<br />
<p>In the rapamycin system the gene of interest is under the control of a minimal promoter. The gene's transcription rate is regulated by two proteins that consist of a drug binding domain and either a DNA binding domain or an activation domain. When rapamycin is added both drug binding domains bind to it, consequently joining the activation domain with the DNA binding domain, resulting in a functional transcription factor, which activates the gene of interest. Instead of rapamycin a rapamycin analogue (rapalogue), which is a 1000-fold less imunosupressive than rapamycin, but activates the inducible system like rapamycin, is usually used as the inducer. (Pollock et al., 2002) </p><br />
<br />
<!-- table --><br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<br />
<th> Regulating vector </th><br />
<br />
<th> HetAct </th><br />
<br />
</tr><br />
</thead><br />
<br />
<br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal"> DNA sequence </td><br />
<td class="normal"> ZFHD </td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal"> Constructs </td><br />
<td class="normal"> ZFHD_pMIN_TAL-A:KRAB </td><br />
</tr> <br />
<br />
<br />
<tr class="normal"><td class="normal"> </td><br />
<td class="normal"> ZFHD_pMIN_TAL-B:VP16</td><br />
</tr> <br />
<br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
</br><br />
<h3>Induction of cells</h3><br />
<ol><br />
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li><br />
<li>Two hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li><br />
</ol><br />
</br><br />
<br />
<table class="normal" style="font-size:90%; width:90%; text-align:center;"><br />
<thead class="normal"><br />
<tr class="normal"><br />
<th>Inductor</th><br />
<th>Stock solution (solvent)</th><br />
<th>Dilution (solvent)</th><br />
<th>Concentration in cell medium (% solvent)</th><br />
</tr><br />
</thead><br />
<tbody class="normal"><br />
<tr class="normal"><td class="normal">Rapalogue (AP21967)</td><br />
<td class="normal">1 mM (100% ethanol)</td><br />
<td class="normal">10 µM (1% ethanol)</td><br />
<td class="normal">1 µM (0,1% ethanol)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Doxycyclin</td><br />
<td class="normal">1 g/L (MQ)</td><br />
<td class="normal">10 mg/L (MQ)</td><br />
<td class="normal">1 mg/L (MQ)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Pristinamycin</td><br />
<td class="normal">50 g/L (100% DMSO)</td><br />
<td class="normal">20 mg/L (1% DMSO)</td><br />
<td class="normal">2 mg/L (0,1% DMSO)</td><br />
</tr> <br />
<br />
<tr class="normal"><td class="normal">Erythromycin</td><br />
<td class="normal">50 g/L (100% ethanol)</td><br />
<td class="normal">20 mg/L (1% ethanol)</td><br />
<td class="normal">2 mg/L (0,1% ethanol)</td><br />
</tr> <br />
</tbody><br />
</table> <br />
<br />
<br />
<br />
<!-- ----------------------------------------------------------------- --><br />
</br><br/><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="effectors"> Effectors </a></h2><br />
<h3>Biological assay-anakinra </h3><br />
<ol><br />
<li>HEK293T cells, seeded in 6-well plate, were transfected with anakinra downstream of constitutive promoter. </li><br />
<li>Transfected cells were incubated for 48 h. </li><br />
<li>To detect anakinra's effect on NF-κB signalling pathway, other HEK293T cells were transfected with plasmid coding for Renilla luciferase and plasmid reporter with NF-κB-inducible firefly luciferase expression. HEK293T cells express IL-1R, so additional transfection with a receptor gene was notneeded. </li><br />
<li>After 24 h, the medium was removed from cells transfected with reporter plasmids and 90 μL of anakinra-producing cells' supernatant was added to these wells. </li><br />
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li><br />
</ol><br />
</br><br />
<br />
<h3> Biological assay-IFN-alpha </h3><br />
<ol><br />
<li>HEK293T cells transfected with eithera plasmid encoding IFN-alpha under the control of a constitutive promoter or an empty vector, and HEK293T cells transfected with the reporter vector were co-cultivated . </li><br />
<li>Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha encoding plasmids. </li><br />
<li>Day after transcfection cells were cultivated into 96-well plate at density 5x104 cells per well.</li><br />
<li>After 24 hours of incubation, dual luciferase reporter assay was performed. </li><br />
</ol><br />
</br><br />
<br />
<h3>ELISA for IFN-alpha</h3><br />
<ol><br />
<li>HEK293T cells where plated on 6 well plates and transfected with a plasmid coding for human IFN-alpha under the control of a constitutive promoter or a control plasmid (pcDNA3). </li><br />
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-fluorescence(The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in black 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega ) per well. </li><br />
<li>Fluorescence was measured using an automated plate reader. </li><br />
</ol><br />
</br><br />
<br />
<h3>Plate reader-luminescence (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 96-well plates and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two to three days of cultivation. </li><br />
<li>After a maximum of 6 days after transfection, cells were lysed with 25 µL of 1x Passive lysis buffer (Promega). </li><br />
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li><br />
</ol><br />
</br><br />
<br />
<br />
<h3>Plate reader-absorbance (The Switch) </h3> <br />
<ol><br />
<li>HEK293T cells were seeded in white 24-well plates and transfected with plasmids encoding the positive feedback loop switch and 10x[TALB + TALC] operator_CMV promoter_fLuciferase reporter plasmid and 10x[TALA + TALC]operator_CMV promoter_SEAP plasmid. Plasmids and amounts used for transfection are listed in Figure legends.</li><br />
<li>Media supplemented with inducer or without inducer were changed after two days of cultivation.</li><br />
<li>Two and seven days after transfection the growth medium was collected and SEAP QUANTIBlue substrate was added. After 15 minutes incubation at 37°C the absorbance was measured at 630 nm. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<h2><a name="microscopy"> Microscopy </a></h2><br />
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p><br />
</br><br />
<br />
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3><br />
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li><br />
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell growth (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng mCitirne (pPCMV-mCitrine) (for transfection control). </li><br />
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li><br />
<li>After 5 days of cultivation, a cell cultures were imaged. </li><br />
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite mCitrine reporter protein. Fluorescence emission was detected at 520-560 nm for mCitrine. Bright field images were used to visualize the number of cells. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-cell count (Safety mechanisms) </h3><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30). </li><br />
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the figure legend. </li><br />
<li>After incubation the cells were resuspended by pipetting. </li><br />
<li>Cells suspensions were then mixed with trypan blue. </li><br />
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-detection of reporter proteins (The Switch) </h3><br />
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p><br />
<ol><br />
<li>HEK293T cells were seeded in an 8-well microscope chamber or 12-well plate and transfected with plasmids encoding the switch. Plasmids and amounts used for transfection are listed in Figure legends. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. Inducers and their concentration are described in Figure legends. </li><br />
<li>Media supplemented with inducer or without inducer were changed after two or three days of cultivation. </li><br />
<li>Images of cells expressing reporters were taken two days after transfection and then each day for 5 days. </li><br />
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-alginate degradation (Microencapsulation) </h3><br />
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation uppon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p><br />
<ol><br />
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li><br />
<li>20 µL of 1 mg/mL FITC-dextran were added into well. </li><br />
<li>After the dye was evenly distributed throughout the suspension, 8 µL of Sphingobacterium multivorum alginate lyase were added. </li><br />
<li>The microscope was set to capture images every 20 seconds. </li><br />
<li>Screenshots were collected for at least 15 minutes. </li><br />
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li><br />
</ol><br />
</br><br />
<br />
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3><br />
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both living and dead cells, while 7-AAD stains dead cells only.</p><br />
<ol><br />
<li>Encapsulated cells were grown in DMEM culture medium supplemented with 10% FBS. </li><br />
<li>200 µL of the microcapsule suspension was collected and alginate-PLL capsules were seeded into an 8-well microscope chamber. </li><br />
<li>5 µL of 7-AAD and 1 µL of Hoechst stain were added to one well. </li><br />
<li>Encapsulated cells were protected from direct light and stained for 30 min at 37 °C. </li><br />
<li>A 405-nm diode laser was used to excite Hoechst and a 543-nm line of HeNe laser was used to excite 7-AAD. </li><br />
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="flowcytometry"> Flow cytometry </a></h2><br />
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p><br />
</br><br />
<br />
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labelled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with phosphatidylserine exposed on their surface.</p><br />
<ol><br />
<li>HEK293 cells were seeded in 12-well plates. </li><br />
<li>Cells were transfected with pCMV-mGMK_TK30 and treated with ganciclovir. Concentrations of ganciclovir and plasmids are indicated in figure legends. </li><br />
<li>After incubation the cells were washed with PBS buffer and resuspended by pipetting. </li><br />
<li>Cells were pelleted with centrifugation at 1200 rpm. </li><br />
<li>The cell pellet was washed in 1x Annexin Binding Buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). </li><br />
<li>The pellet was then resuspended in 1x Annexin Binding Buffer and PE-Annexin V (5 µl per 100 μl cell suspension) was added. </li><br />
<li>Samples were incubated for 20 minutes in the dark at room temperature and then immediately analyzed with a flow cytometer. </li><br />
<li>Along with site and forward scatter, the signal in the FL2 channel (540-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3><br />
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p><br />
<ol><br />
<li>HEK293T cells seeded in 12-well plates were transfected with plasmids expressing MICA (pPCMV-MICA_pcDNA3) and/or a blue fluorescent protein (BFP) (pPCMV-BFP). BFP was used to discriminate between HEK293T and NK-92 cells. </li><br />
<li>Two days after transfection cells were mixed with NK-92 cells in different ratios (1:1, 1:5, 1:10) and incubated for 4 hours at 37 °C in culture medium consisting of RPMI, 20% FBS and hIL-2 (100 U/ml). </li><br />
<li>After incubation of HEK293T with NK-92 cells, cells were treated with propidium iodide. </li><br />
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded. </li><br />
</ol><br />
</br><br />
<br />
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3><br />
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p><br />
<ol><br />
<li>HEK293T cells were seeded in a 12 or 24-well plate and transfected with plasmids encoding the switch. </li><br />
<li>Two hours after transfection, media was changed and cells were stimulated with inducers. </li><br />
<li>Medium with inducer or without the inducer was changed after two days of cultivation. </li><br />
<li>Cells were collected at different time points (2 days after induction and then 3 days after the second media change). </li><br />
<li>Cells were washed and resuspended in PBS buffer. </li><br />
<li>A 405 nm diode laser was used to excite tagBFP and a 488-nm diode laser was used for mCitirne. </li><br />
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li><br />
</ol><br />
</br><br />
<br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="microencapsulation"> Microencapsulation </a></h2><br />
<h3>Cell preparation for encapsulation</h3><br />
<ol><br />
<li>HEK 293T cells were seeded 5x10⁵ per 10 cm cell culture dish (3 per encapsulation) and grown in DMEM medium supplemented with 10 % FBS. </li><br />
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection). </li><br />
<li>The medium was removed the next day.. Transfected cells were detached using 3 mL of trypsin solution and centrifuged after the addition of fresh medium to inactivate the trypsin. </li><br />
<li>Supernatant was removed and cells were resuspended in 15 mL DMEM with 10% FBS. </li><br />
<li>Cells were counted using Countess automated cell counter (Invitrogen). </li><br />
<li>HEK 293T cells were again centrifuged and supernatant was removed. </li><br />
<li>Cells were resuspended in 2 mL of pre-warmed MOPS buffer. </li><br />
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li><br />
</ol><br />
</br><br />
<br />
<h3>Encapsulation</h3><br />
<ol><br />
<li>The encapsulator was equipped with a 200-µm nozzle. </li><br />
<li>The reactor vessel was filled with 225 mL 100 mM CaCl2. </li><br />
<li>Cell-alginate mixture was transferred to a 20 mL syringe with a Luer lock. </li><br />
<li>The syringe was connected to the bead producing unit (BPU). </li><br />
<li>Microcapsules were produced at a flow rate of 12-14 units, vibration frequency 1030-1100 Hz and voltage for bead dispersion 900-1300 V. </li><br />
<li>Polymerisation lasted for 10 min. </li><br />
<li>The polymerization solution was drained and 75 mL of 0,05% poly-L-lysine (PLL) solution was added. </li><br />
<li>Beads were incubated in PLL solution for 10 minutes. </li><br />
<li>The PLL solution was removed and beads were washed twice (for 1 and for 5 min) with 150 mL of MOPS buffer. </li><br />
<li>100 mL of 0,03% alginate was added and beads were incubated for 10 min. </li><br />
<li>Alginate solution was drained and beads were washed once with 150 mL of MOPS buffer for 1 min. </li><br />
<li>150 mL of depolymerization solution was added for 10 min. </li><br />
<li>Depolymerization solution was removed and capsules were resuspended in 150 mL MOPS and collected in a bead collection flask. </li><br />
<li>MOPS was removed and microcapsules were transferred to T-75 with 10 mL DMEM, 10% FBS media supplemented with penicillin and streptomycin. </li><br />
</ol><br />
<br />
<br/><br />
<p><b>Buffers and solutions</b><br />
<br/><br />
<br><b>10 mM MOPS buffer</b> (pH = 7,2)<br />
<br><b>MOPS buffer with NaCl</b> (pH = 7,2): 10 mM MOPS, 0,85% NaCl<br />
<br><b>Polymerisation solution</b> (pH = 7,2): 10 mM MOPS, 100 mM CaCl2<br />
<br><b>Depolymerisation solution</b> (pH = 7,2): 10 mM MOPS, 50 mM Na3-citrate, 0,45% NaCl<br />
<br><b>0,05% poly-L-lysine</b> (15-30 kDa) in MOPS buffer (pH = 7,3) <br />
<br><b>0,03% alginate</b> in MOPS buffer (pH = 7,2)<br />
<br><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2)</p><br />
</br><br />
<br />
<p><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/Notebook#naslov">Back to top</a></p><br />
<br />
<h2><a name="proteindetection"> Protein detection </a></h2><br />
<h3>SDS-PAGE</h3><br />
<ol><br />
<li>Samples were loaded on a 12% acrylamide gel and ran at a constant voltage (200 V) for 1 h. </li><br />
<li>Proteins were then blotted on a nitrocellulose membrane at a constant current (350 mA) for 1 h. </li><br />
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li><br />
</ol><br />
</br><br />
<br />
<h3>Immunodetection </h3><br />
<ol><br />
<li>All proteins to be detected had a Myc tag at the C-terminus. </li><br />
<li>The membrane was incubated with primary antibodies (rabbit anti-Myc diluted 1:500) overnight at 4 °C and 150 rpm. Membrane was washed. </li><br />
<li>Washing in wash buffer three times for 5 min each.</li><br />
<li>Membrane was incubated with secondary antibodies (anti-rabbit secondary antibodies, conjugated with HRP, diluted 1:3000) for 45 min at room temperature and 150 rpm. </li><br />
<li>HRP activity was detected by addition of SuperSignal West Femto or Pico Substrate (Thermo Scientific). Images were captured with Syngene G:Box chemiluminescent imaging system. </li><br />
</ol><br />
<p><b>Buffers and solutions </b></p><br />
<p><b>Wash buffer</b>: 1x PBS, 0,01% (v/v) Tween 20</p><br />
</br><br />
<br />
</p><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Clackson, T. (2000) Regulated gene expression systems. <i> Gene ther.</i> <b>7</b>, 120–125.<br/><br/><br />
Deuschle, U., Meyer, W.K. and Thiesen, R. (1995) Tetracycline-reversible silencing of eukaryotic promoters. <i>Mol. Cell. Biol. </i> <b>15</b>, 1907–1914.<br/><br/><br />
Kramer, B.P., Fischer, C. and Fussenegger, M. (2004) BioLogic gates enable logical transcription control in mammalian cells. <i> Biotech. Bioeng.</i> <b>87</b>, 478–484.<br/><br/><br />
Pollock, R. and Clackson, T. (2002) Dimerizer-regulated gene expression. <i> Curr. Opin. Biotech. </i> <b>13</b>, 459–467.<br/><br/><br />
Gibson, D.G., Young, L., Chuang, R., Venter J.C., Hutchison III, C. A. and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature methods.</i> <b>6</b>, 343–345.<br />
</p><br />
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<br />
<br/><br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'>Lablog >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T16:08:57Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALs”>TAL regulators</a> (<a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004”>TALA</a>, <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006”>TALB</a> and <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005”>TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href=”https://2012.igem.org/Team:Slovenia/Parts#reporters”>reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href=”http://partsregistry.org/Part:BBa_K323214”>NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href=”http://partsregistry.org/Part:BBa_K782007”>NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<h3>Designed repressors</h3><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009”>KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALeffectors”>TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011”>NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALactivators”>TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrin under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter.</p><br />
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<b>Figure 9. TAL-based transcriptional activator is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results thus demonstrate that TAL-based logic can be used also in this multicellular animal, which could be used as a model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TeamCollaborations
Team:Slovenia/TeamCollaborations
2012-10-26T16:03:40Z
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
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<br />
<h1>Collaborations</h1><br />
<p><br />
We collaborated with <a href="https://2012.igem.org/Team:Evry">team Evry</a> to investigate if TAL-based transcriptional regulators are functional in cells of amphibians and in multicellular animals. Frog oocytes and embryos transfected with plasmids containing mCitrine reporter under the minimal promoter and an upstream TAL-operator exhibited fluorescence when it was cotransfected with TAL-VP16 activator. This fluorescence was absent in cells and animals that were transfected only with the mCitrine reporter (described in the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators">Switch section</a>). This demonstrates that TAL based genetic logic could be used to regulate complex properties in multicellular organisms, which has many important implications for the potential therapeutic use.<br />
</p><br />
<br />
<p>We provided parts that were not included in the distribution to the <a href="https://2012.igem.org/Team:Warsaw">Warsaw team</a>.</p><br />
<br />
<p>We provided additional information about the part development to <a href="https://2012.igem.org/Team:Peking">Peking team</a>.</p><br />
<br />
<p>We were pleased when the <a href="https://2012.igem.org/Team:Wageningen_UR">Wageningen team</a> sent us a mail after the European jamboree that they were amazed by our project and that they introduced features of our pharmacokinetic model into their project.</p><br />
<br />
<p>We participated in the survey of <a href="https://2012.igem.org/Team:TU_Munich">TU Munich team</a>.</p><br />
<br />
<p>We had discussions with a member of <a href="https://2012.igem.org/Team:St_Andrews">St. Andrews team</a> that visited us. We welcome all other visitors as well.</p><br />
<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/TeamGallery'>Gallery >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T16:02:31Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br/><br />
<br />
<h1> TAL-based transcriptional regulators</h1><br />
<br />
<table class="summary"><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALs”>TAL regulators</a> (<a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004”>TALA</a>, <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006”>TALB</a> and <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005”>TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href=”https://2012.igem.org/Team:Slovenia/Parts#reporters”>reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href=”http://partsregistry.org/Part:BBa_K323214”>NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href=”http://partsregistry.org/Part:BBa_K782007”>NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009”>KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALeffectors”>TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011”>NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALactivators”>TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrin under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter.</p><br />
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<b>Figure 9. TAL-based transcriptional activator was active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results thus demonstrate that TAL-based logic can be used also in this multicellular animal, which could be used as a model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T16:02:01Z
<p>Dusanv: </p>
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
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<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<table class="summary"><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALs”>TAL regulators</a> (<a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004”>TALA</a>, <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006”>TALB</a> and <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005”>TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href=”https://2012.igem.org/Team:Slovenia/Parts#reporters”>reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href=”http://partsregistry.org/Part:BBa_K323214”>NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href=”http://partsregistry.org/Part:BBa_K782007”>NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009”>KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALeffectors”>TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011”>NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALactivators”>TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrin under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter.</p><br />
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<b>Figure 9. TAL-based transcriptional activator was is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results thus demonstrate that TAL-based logic can be used also in this multicellular animal, which could be used as a model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
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Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
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Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
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Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
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Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
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Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/File:Svn12_evryfrogs.png
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2012-10-26T16:00:15Z
<p>Dusanv: uploaded a new version of &quot;File:Svn12 evryfrogs.png&quot;</p>
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators
Team:Slovenia/TheSwitchDesignedTALregulators
2012-10-26T15:59:21Z
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
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<h1> TAL-based transcriptional regulators</h1><br />
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<p>We created several <b>TAL repressors</b> by fusions of the <b>KRAB repression domain</b> to different positions relative tothe TAL DNA-binding domain and <b>reporter plasmids with their respective binding sites (operators). </b></p><br />
<p>We created <b>several TAL activator constructs</b> by C-terminal fusion of the <b>VP16 domain with TAL DNA-binding domains</b> and reporter plasmids with their respective operators. </p><br />
<p>We <b>improved and characterized the NicTAL DNA-binding domain</b> (deposited by the iGEM2010 team Slovenia) by adding the missing subdomain of the protein and created a designed repressor and activator. </p><br />
<p>TAL:KRAB fusions exhibited over <b>90% repression</b> of reporter gene expression <b>regardless of the position of the KRAB domain. </b> </p><br />
<p><b>Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness</b> and were <b>activated over 1500-fold</b> by TAL:VP16 fusions. </p><br />
<p>Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches. </p><br />
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<h2>Designed TAL transcriptional regulators </h2><br />
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<p>For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighboring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. <b>Transcription activator like <a href ="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">(TAL) effectors</a></b> are bacterial plant pathogen transcription factors that bind to DNA by <b>recognizing a specific DNA sequence in which each base pair binds to a single tandem repeat in the TAL DNA-binding domain</b> (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figure 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This <b>modularity</b> of TAL effector binding domains therefore makes them a <b>perfect tool</b> to target specific DNA sequences. </p><br />
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<p><b>Figure 1.The structure of TAL effectors. </b> (A) Schematic representation of TAL effector structure and its DNA-binding domain (red), containing multiple 34 aminoacid tandem repeats with RVDs at the 12th and 13th residue (Scholze and Boch, 2011). (B) 3D structure of a TAL effector. </p><br />
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<p><b>Figure 2.Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B). </b></p><br />
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<p>TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011; Garg et al., 2012; Cong et al., 2012) have also designed TAL effectors for <b>specific gene activation,</b> by fusing them with either <b>the Herpes simplex virus VP16 activation domain</b> or its <b>tetrameric derivative VP64</b> (Figure 2). After we already initiated the iGEM 2012 project, <b>TAL repressors</b> were reported by Garg et al., and Cong et al.,who created TAL effectors fused with <b>the KRAB or SID transcriptional repression domain. </b> </p><br />
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<h2>Results</h2><br />
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<b>Figure 3. Schematic representation of tested plasmids. </b> (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).<br />
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<p>The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterized three functional <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALs”>TAL regulators</a> (<a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004”>TALA</a>, <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006”>TALB</a> and <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782005”>TALD</a>) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To assess the activity of designed TAL regulators, we also designed <a href=”https://2012.igem.org/Team:Slovenia/Parts#reporters”>reporter plasmids</a>, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C). </p><br />
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<p>In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named <a href=”http://partsregistry.org/Part:BBa_K323214”>NicTAL</a>, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the <a href=”http://partsregistry.org/Part:BBa_K782007”>NicTAL-DNA binding domain</a> and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators. </p><br />
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<h3>Designed repressors</h3><br />
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<p>We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs offour different TAL domains exhibited <b>over 90% repression of the reporter plasmid</b> (with the exception of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782009”>KRAB:TALD</a>). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that <b>the position of the effector (regulator) domain on either the N- or C-terminus or both does not influence the binding and repression ability of the designed TAL repressors. </b> All further experiments were performed with <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALeffectors”>TAL:KRAB</a> fusions. </p><br />
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<tr class="inliner"><td class="inliner"><b>Figure 4. Schematic representation of repression experiments. </b> (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing. <br />
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<b>Figure 5. TAL repressors potently inhibit expression of reporter genes. </b> HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results. <br />
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<p>Results show that the previously non-functional NicTAL10:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An <b>excellent repression ability of <a href=”http://partsregistry.org/wiki/index.php?title=Part:BBa_K782011”>NicTAL12:KRAB</a> (the improved version by the 2012 team) </b> was observed. The NicTAL12 repressor was further characterized by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the <b>maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. </b> In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites. </p><br />
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<b>Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. </b> HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment was executed in threebiological replicates and repeated three times with similar results. <br />
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<b>Figure 7. Schematic representation of activation experiments. </b> (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene. <br />
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<p>Due to the mechanism of action of the VP16 domain and our previous results with TAL:KRAB repressors (Figure 5, position of the effector domain does not influence repression); we decided to test only the C-terminal variant of the <a href=”https://2012.igem.org/Team:Slovenia/Parts#TALactivators”>TAL:VP16 fusion</a> (Figure 3A). Both tested TAL activators exhibited <b>over 1500-fold activation</b> of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we confirmed that <b>the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness</b> - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed. </p><br />
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<b>Figure 8. TAL activators strongly activate the reporter gene expression. </b> The number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with TAL activator constructs under the CMV promoter (different quantities, for ratios see the x axis), and mCitrine reporter plasmids (Figure 3B) containing 10 copies of binding sites for the designated TAL activator upstream of a minimal promoter (50 ng). Along with the tested constructs we transfected cells with 20 ng of mCherry fluorescent protein under the CMV promoter as transfection control. Fluorescence was measured three days post-transfection. All experiments were executed in 3 biological replicates and repeated three times with similar results. <br />
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<p>In collaboration with iGEM <a href="https://2012.igem.org/Team:Evry/FrenchFrog">team Evry</a> we tested the ability of TAL regulators to function in cells of amphibians. We selected a reporter plasmid with mCitrin under the operator for TAL VP16 activator in the presence and absence of the TAL activator. Only cells of animals transfected with both plasmids exhibited fluorescence, which was absent in cells transfected only with reporter.</p><br />
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<b>Figure 9. TAL-based transcriptional activator was is active in the multicellular organism.</b> Reporter plasmid (10x[TALA] pMIN mCitrine, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a>) was injected into <i>Xaenopus laevis</i> embryo (2.3 nl of plasmid at concentration 100 ng/µl) in the presence or absence of a plasmid with TAL activator (pCMV-TALAVP16, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a>). Fluorescence of mCitrin and rhodamine, which was used with plasmid for injection control was observed under the microscope as described <a href="https://2012.igem.org/Team:Evry/Protocols">https://2012.igem.org/Team:Evry/Protocols</a>.<br />
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<p>Our joint results thus demonstrate that TAL-based logic can be used also in this multicellular animal, which could be used as a model to study complex synthetic regulatory devices in the multicellular environment.</p><br />
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<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. <i>Science</i> <b>335</b>, 720-723.<br />
<br/><br/><br />
Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. <i>Nucleic Acids Res.</i> <b>40</b>, 7584-95.<br />
<br/><br/><br />
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. <i>Nat Commun.</i>3, 968.<br/><br/><br />
Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. <i>Science</i> <b>335</b>, 716-719.<br />
<br/><br/><br />
Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. <i>Nat. Biotechnol.</i> <b>29</b>, 143-148.<br />
<br/><br/><br />
Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. <i>Nat. Biotechnol.</i> <b>29</b>, 697–698.<br />
<br/><br/><br />
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. <i>Curr. Opin. Microbiol.</i> <b>14</b>, 47-53. <br />
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Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. <i>Nat. Biotechnol.</i> <b>29</b>, 149-153.<br />
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<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'>Mutual repressor switch >></a><br />
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Dusanv
http://2012.igem.org/File:Svn12_evryfrogs.png
File:Svn12 evryfrogs.png
2012-10-26T15:50:07Z
<p>Dusanv: </p>
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Dusanv
http://2012.igem.org/Team:Slovenia/TeamCollaborations
Team:Slovenia/TeamCollaborations
2012-10-26T14:58:54Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
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<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
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<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br />
<h1>Collaborations</h1><br />
<p><br />
We collaborated with <a href="https://2012.igem.org/Team:Evry">team Evry</a> to investigate if TAL-based transcriptional regulators are functional in cells of amphibians and in multicellular animals. Frog oocytes and embryos transfected with plasmids containing mCitrine reporter under the minimal promoter and an upstream TAL-operator exhibited fluorescence when it was cotransfected with TAL-VP16 activator. This fluorescence was absent in cells and animals that were transfected only with the mCitrine reporter (described in the <a href="https://2012.igem.org/Team:Slovenia/TheSwitch">Switch section</a>). This demonstrates that TAL based genetic logic could be used to regulate complex properties in multicellular organisms, which has many important implications for the potential therapeutic use.<br />
</p><br />
<br />
<p>We provided parts that were not included in the distribution to the <a href="https://2012.igem.org/Team:Warsaw">Warsaw team</a>.</p><br />
<br />
<p>We provided additional information about the part development to <a href="https://2012.igem.org/Team:Peking">Peking team</a>.</p><br />
<br />
<p>We were pleased when the <a href="https://2012.igem.org/Team:Wageningen_UR">Wageningen team</a> sent us a mail after the European jamboree that they were amazed by our project and that they introduced features of our pharmacokinetic model into their project.</p><br />
<br />
<p>We participated in the survey of <a href="https://2012.igem.org/Team:TU_Munich">TU Munich team</a>.</p><br />
<br />
<p>We had discussions with a member of <a href="https://2012.igem.org/Team:St_Andrews">St. Andrews team</a> that visited us. We welcome all other visitors as well.</p><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TeamCollaborations
Team:Slovenia/TeamCollaborations
2012-10-26T14:57:27Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
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</a><br />
<br />
<h1>Collaborations</h1><br />
<p><br />
We collaborated with <a href="https://2012.igem.org/Team:Evry">team Evry</a> to investigate if TAL-based transcriptional regulators are functional in cells of amphibians and in multicellular animals. Frog oocytes and embryos transfected with plasmids containing mCitrine reporter under the minimal promoter and an upstream TAL-operator exhibited fluorescence when it was cotransfected with TAL-VP16 activator. This fluorescence was absent in cells and animals that were transfected only with the mCitrine reporter (described in <a href="https://2012.igem.org/Team:Slovenia/TheSwitch">the Switch section</a>). This demonstrates that TAL based genetic logic could be used to regulate complex properties in multicellular organisms, which has many important implications for the potential therapeutic use.<br />
</p><br />
<br />
<p>We provided parts that were not included in the distribution to the <a href="https://2012.igem.org/Team:Warsaw">Warsaw team</a>.</p><br />
<br />
<p>We provided additional information about the part development to <a href="https://2012.igem.org/Team:Peking">Peking team</a>.</p><br />
<br />
<p>We were pleased when the <a href="https://2012.igem.org/Team:Wageningen_UR">Wageningen team</a> sent us a mail after the European jamboree that they were amazed by our project and that they introduced features of our pharmacokinetic model into their project.</p><br />
<br />
<p>We participated in the survey of <a href="https://2012.igem.org/Team:TU_Munich">TU Munich team</a>.</p><br />
<br />
<p>We had discussions with a member of <a href="https://2012.igem.org/Team:St_Andrews">St. Andrews team</a> that visited us. We welcome all other visitors as well.</p><br />
<br />
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Next: <a href='https://2012.igem.org/Team:Slovenia/TeamGallery'>Gallery >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TeamCollaborations
Team:Slovenia/TeamCollaborations
2012-10-26T14:53:59Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
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<br />
<h1>Collaborations</h1><br />
<p><br />
We collaborated with <a href="https://2012.igem.org/Team:Evry">team Evry</a> to investigate if TAL-based transcriptional regulators are functional in cells of amphibians and in multicellular animals. Frog oocytes and embryos transfected with plasmids containing mCitrine reporter under the minimal promoter and an upstream TAL-operator exhibited fluorescence when it was cotransfected with TAL-VP16 activator. This fluorescence was absent in cells and animals that were transfected only with the mCitrine reporter (described in the Switch section). This demonstrates that TAL based genetic logic could be used to regulate complex properties in multicellular organisms, which has many important implications for the potential therapeutic use.<br />
</p><br />
<br />
<p>We provided parts that were not included in the distribution to the <a href="https://2012.igem.org/Team:Warsaw">Warsaw team</a>.</p><br />
<br />
<p>We provided additional information about the part development to <a href="https://2012.igem.org/Team:Peking">Peking team</a>.</p><br />
<br />
<p>We were pleased when the <a href="https://2012.igem.org/Team:Wageningen_UR">Wageningen team</a> sent us a mail after the European jamboree that they were amazed by our project and that they introduced features of our pharmacokinetic model into their project.</p><br />
<br />
<p>We participated in the survey of <a href="https://2012.igem.org/Team:TU_Munich">TU Munich team</a>.</p><br />
<br />
<p>We had discussions with a member of <a href="https://2012.igem.org/Team:St_Andrews">St. Andrews team</a> that visited us. We welcome all other visitors as well.</p><br />
<br />
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Dusanv
http://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease
Team:Slovenia/ImplementationIschaemicHeartDisease
2012-10-26T02:57:52Z
<p>Dusanv: </p>
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<h1>Ischaemic heart disease</h1><br />
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<p>We designed a device for the therapy of myocardial ischaemia, composed of microencapsulated mammalian cells that include a genetic bistable toggle switch with a positive feedback loop, where in the first state cells produce anakinra as the anti-inflammatory effector and in the second state they produce a stoichiometric amount of the vascular endothelial growth factor (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782061">VEGF</a>) and platelet-derived growth factor B (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782061">PDGF-BB</a>) to promote angiogenesis in the damaged tissue.</p><br />
<p>A pharmacokinetic model demonstrated that implantation of the device into the injured heart tissue results in a high level of anakinra concentration within the affected tissue, while the systemic level of anakinra is negligible, preventing systemic immunosuppression.</p><br />
<p>We demonstrated that the production level of anakinra by engineered cells is sufficient for the therapeutic implementation of microencapsulated cells for this indication.</p><br />
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<tr class="invisible"><td class="invisible"><b>Figure 1. Regulated release of therapeutic proteins into the tissue.</b> <br />
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<b>Figure 2. Scheme of the constructs for the regulated therapy of ischaemia with IL-1Ra (IL-1 receptor antagonist, anakinra) and VEGF/PDGF-BB to suppress inflammation and promote angiogenesis, respectively.</b><br />
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<h3>Ischaemic heart disease</h3><br />
<p>Ischaemic heart disease is characterized by a reduced blood supply to the heart muscle, usually due to coronary artery disease (atherosclerosis of the coronary arteries). Ischaemic heart disease (which includes myocardial infarction, angina pectoris and heart failure when preceded by myocardial infarction) is <b>the leading cause of mortality in most Western countries</b>. Menzin et al. reported a total first-year cost averaged of 32,345 $ and as much as 61% of these costs were due to rehospitalisation (Menzin et al., 2008). Furthermore, survivors of acute myocardial infarction remain at high risk of death in the years after the event (Grothusen et al., 2012).</p><br />
<h4>Current therapy and research</h4><br />
<p><b>Acute myocardial infarction</b> due to total occlusion of a coronary artery is treated with early reperfusion strategies to minimize the developing ischaemic myocardial damage. Current guidelines recommend interventional treatment via <b>percutaneous transluminal coronary angioplasty (PTCA)</b> of the occluded coronary artery followed by a stent implantation in the setting of acute myocardial infarction. In case of PTCA contraindications, <b>operative revascularization therapy CABG</b> (coronary artery bypass graft surgery) is recommended. However, many studies have shown a negative effect of reperfusion, resulting in additional myocardial injury due to activation of an inflammatory response (Grothusen et al., 2012; Abbate et al., 2008).</p><br />
<p>Interleukin-1a and b are one of the most important inflammatory cytokines which are responsible for leukocyte chemotaxis, macrophage activation, reactive oxygen species formation, endothelial dysfunction, and cardiomyocyte apoptosis. Thus, <b>inhibition of IL-1 receptor complex activation</b> can present a new important treatment target (Grothusen et al., 2012). Furthermore, recent studies have shown the <b>cardioprotective properties of anakinra</b>, which is a nonglycosylated, human recombinant competitive inhibitor of IL-1a and b signaling through binding to the IL-1 receptor complex (Abbate et al., 2008).</p><br />
<p>There is also experimental evidence of the important <b>regenerating role of vascular endothelial growth factor (VEGF)</b> after acute myocardial infarction (Banfi et al., 2012; Zhang et al., 2008). After binding to its receptor VEGFR1 and VEGFR2 it supports angiogenesis, inhibits endothelial cell apoptosis, promotes endothelial cell proliferation, and restores heart function. Although therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy, many clinical trials have thus far failed to show efficiency. One of the most likely explanations for this discrepancy is that VEGF induces growth of dysfunctional vessels, if expressed outside of a narrow dosage window (Banfi et al., 2012). Banfi et al. confirmed the hypothesis that <b>co-delivery of platelet-derived growth factor-BB (PDGF-BB)</b>, which recruits pericytes, induced <b>normal angiogenesis</b> in skeletal muscle irrespective of VEGF levels. It was also shown that coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Secondly, a challenge has been that with the conventional gene transfer vectors, the growth factor concentration in target tissues had not reached sufficient levels or had not persisted long enough for triggering relevant vascular growth (Zhang et al., 2008). Cell transplantation strategies emerged as a promising approach to overcome this issue.</p><br />
<p>Zhang et al. reported the first transplantation of microencapsulated engineered xenogeneic CHO cells in post-infarction myocardium, which showed that the supplementation of VEGF from implanted xenogeneic cells could foster the formation of arterial collaterals, improve myocardial perfusion and thus also promote the regeneration of damaged myocardium augmented angiogenesis and improved heart function (Zhang et al., 2008).</p><br />
<h4>Implementation of the microencapsulated engineered cellular device for the therapy of ischaemic heart disease</h4><br />
<p>With our biological delivery system for biopharmaceuticals, we aim to improve the therapy of ischaemic heart disease by delivering multiple biopharmaceuticals in different time periods controlled by a physician. The encapsulated cells would be locally administered with intracardial injections via ventriculography, or intraoperatively in the proximity of the myocardial damage (Figure 1). <b>In the first phase</b>, after myocardial damage has occurred and reperfusion injury is expected, production of <b>anakinra</b> could halt the inflammatory response. <b>In the second phase</b>, the therapy would switch to the production of <b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782061">VEGF and PDGF-BB</a></b> at stoichiometric concentrations, which could enhance the production of normal, robust vessels (Figure 2). After the therapeutic effect is reached, we could discontinue the synthesis of growth factors through the ability to control the viability of encapsulated allogenic cells. This could prevent the negative side effects of over-production of vascular growth factors (haemangioma formation) and contribute a great deal to biosafety. Because of the local application, we anticipated that systemic effects of anakinra, VEGF and PDGF would be negligible and tested this hypothesis using a pharmacokinetic model. This strategy of treatment could prevent the development of chronic heart failure, which is one of the most common complications after myocardial infarction.</p><br />
<h2>Results</h2><br />
<h3>Pharmacokinetic modeling of the distribution of anakinra in the local antiinflammatory therapy</h3><br />
<p>We prepared a pharmacokinetic model for the local therapy of ischaemic heart disease with anakinra. This model (described in details in the <a href="https://2012.igem.org/Team:Slovenia/Modeling">Modeling</a> section) demonstrates achievement of the local therapeutic concentrations of anakinra, while the systemic concentration remains negligible in contrast to systemic application of anti-inflammatory therapy. The serum half life of anakinra is very low so it needs to be administered daily for systemic application, but implementation of encapsulated cells with a constant production rate of anakinra would circumvent this.</p><br />
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<b>Figure 3. Distribution of anakinra in different tissues and organs based on the pharmacokinetic model of the local therapy of ischaemic heart disease by microencapsulated engineered cells.</b> More intense (saturated) red colors indicate higher concentrations, while low concentrations are closer to transparent/white colors. The model demonstrates that anakinra is highly localized at the place of administration of microcapsules.<br />
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<h3>Detection of biological activity of produced anakinra</h3><br />
<p>IL-1β signals through binding of IL-1 receptor type 1 (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP), which leads to a juxtaposition of the intracellular TIR domains of IL-1R1 and IL-1RAcP. The activation of the receptor complex triggers intracellular signaling which results in the activation of the transcription factor NF-κB and the mitogen-activated protein kinase (MAPK) pathways. Anakinra (IL-1 receptor antagonist; IL-1Ra) is a competitive inhibitor that prevents activation of the IL-1 receptor complex (Wang et al., 2010).</p><br />
<p>We wanted to test the biological activity of anakinra produced in HEK293T. We designed an experiment where the inhibition of IL-1β signaling by anakinra was observed. HEK293T cells were transfected with NF-κB-inducible firefly luciferase reporter plasmid and constitutive <i>Renilla</i> luciferase plasmid for normalization. Transfected cells were preincubated with supernatant from anakinra-producing cells or with recombinant anakinra (rIL-1Ra). These cells were then stimulated with IL-1β and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay (Figure 4). Results show almost complete inhibition of IL-1β signaling with recombinant anakinra as well as with supernatant from anakinra-producing cells.</p><br />
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<img style="width:70%;" src="https://static.igem.org/mediawiki/2012/0/0a/Svn12_implementation_ischemia_fig4.png"></img><br />
<center><p style="width:68%;"><b>Figure 4. Inhibition of IL-1β signaling by anakinra.</b> HEK293T cells, transfected with reporter plasmid, were preincubated with supernatant of HEK293T cells producing anakinra under the control of a constitutive promoter or with recombinant anakinra (rIL-1Ra) (500 ng/mL). These cells were then stimulated with IL-1β (10 ng/mL) and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay.</p></center><br />
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<p>We have calculated the therapeutic concentration of anakinra to be 25 ng/mL, which we derived from the anakinra IC_50 value (Dahlén et al., 2008). Our pharmacokinetic model of microencapsulated cell therapy predicted we would need production of approximately <b>30 µg per day in the target tissue</b>. Considering that in our experiments each cell produced around <b>1*10^-6 µg per day</b> and that the number of cells per capsule is 3000-15000 we calculated that we would need to implant between <b>2000-9000</b> capsules into a patch of ischaemic tissue, which would represent a volume of <b>15 to 75µL.</b> These estimates are very rough and would have to be tested experimentally in the tissue but provide the basis for judging the feasibility of this approach. Additional indication may be also provided by the successful application of the local gene therapy of myocarditis (Lim et al., 2002, Suzuki et al., 2001)</p><br />
<br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Abbate, A., Salloum, F.N., Vecile, E., Das, A., Hoke, N.N., Straino, S., Biondi-Zoccai, G.G., Houser, J.E., Qureshi, I.Z., Ownby, E.D., Gustini, E., Biasucci, L.M., Severino, A., Capogrossi, M.C., Vetrovec, G.W., Crea, F., Baldi, A., Kukreja, R.C., and Dobrina, A. (2008) Anakinra, a recombinant human Interleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction. <i>Circulation</i> <b>17</b>, 2670-2683. <br/><br/><br />
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Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M.J., McDonald, D.M., and Blau, H.M. (2012) Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. <i>FASEB J.</i> <b>26</b>, 2486-2497. <br/><br/><br />
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Dahlén, E., Barchan, K., Herrlander, D., Höjman, P., Karlsson, M., Ljung, L., Andersson, M., et al. 2008. Development of Interleukin-1 Receptor Antagonist Mutants with Enhanced Antagonistic Activity In Vitro and Improved Therapeutic Efficacy in Collagen-Induced Arthritis.<i>J Immunotoxicol</i> <b>5</b>, 189-99. <br/><br/><br />
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Grothusen, C., Hagemann, A., Attmann, T., Braesen, J., Broch, O., Cremer ,J., and Schoettler, J. (2012) Impact of an interleukin-1 receptor antagonist and erythropoietin onexperimental myocardial ischemia/reperfusion injury. <i>Scientific World Journal</i> 737585. Epub 2012 May 2<br/><br/><br />
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Lim BK, Choe SC, Shin JO, Ho SH, Kim JM, Yu SS, Kim S, Jeon ES. (2002) Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis.<i>Circulation</i> <b>105</b>, 1278-81.<br/><br/><br />
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Menzin, J., Wygant, G., Hauch, O., Jackel, J., and Friedman, M. (2008) One-year costs of ischemic heart disease among patients with acute coronary syndromes: findings from a multi-employer claims database. <i>Curr. Med. Res. Opin.</i> <b>24</b>, 461-468.<br/><br/><br />
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Suzuki K, Murtuza B, Smolenski RT, Sammut IA, Suzuki N, Kaneda Y, Yacoub MH. (2001) Overexpression of interleukin-1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis. <i>Circulation</i> <b>104</b>, I308-I3.<br/><br/><br />
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Wang D., Zhang S., Li L., Xi Liu, Mei K. and Wang X. (2010) Structural insights into the assembly and activation of IL-1b with its receptors. <i>Nat. immunol</i> <b>11</b>, 10, 905-912.<br/><br/><br />
Zhang, H., Zhu, S.J., Wang, W., Wei, Y.J., and Hu, S.S. (2008) Transplantation of microencapsulated genetically modified xenogeneic cells augments angiogenesis and improves heart function. <i>Gene Ther.</i> <b>15</b>, 40-48.<br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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<br/><br />
<h2>Impact of switches</h2><br />
<br/><br />
<br />
<p>A Switch is the basic regulatory element that can maintain a selected state even in the absence of a signal. Introduction of several orthogonal bistable switches allows selection of many different states, e.g. 3 bistable switches allow 8 distinct states while <b>1000 different states could be reached with only 10 switches</b>.</p><br />
<p>Each state of our switch is defined by the presence of selected activators and repressors than can in principle regulate any number of selected genes. Therefore with a relatively small number of designed switches we could <b>drive a very complex epigenetic cell program</b> such as cell differentiation. </p><br />
<p>Additionally, switches are the <b>basic building blocks of memory</b>, in analogy to the electronic components. Therefore a set of orthogonal bistable switches can be used to build a biological memory of significantly higher complexity than up to now. </p><br />
<p><b>Scalable biological memory is one of the great challenges of synthetic biology.</b> Memory can be used to design counters, which are extremely useful elements that have, so far, been limited to count up to 3 with no prospects of a significant increase using the same underlying tools. </p><br />
<p>We therefore anticipate that designed TAL-based or in general <b>DNA-binding element-based transcriptional factor logic will play a very important role in the development of synthetic biology. </b></p><br />
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<hr><br />
<b><br />
Next: <a href="https://2012.igem.org/Team:Slovenia/Modeling">Modeling >></a><br />
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Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch
Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch
2012-10-26T02:55:45Z
<p>Dusanv: </p>
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
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<br />
<h1> Positive feedback loop switch</h1><br />
<br />
<table class="summary"><br />
<tr class="summary"><br />
<td class="summary"><br />
<p>We designed an upgraded <b>bistable genetic toggle switch based on orthogonal TAL <br />
repressors and activators</b> which is composed of a pair of mutual repressors and a<br />
pair of activators extending the classical toggle swith with positive feedback loops.</p><br />
<p>Simulations of the positive feedback loop switch demonstrated <b>bistability even at low or no cooperativity. </b></p><br />
<p>Stochastic and deterministic simulations indicate higher robustness in comparison to the mutual repressor switch. </p><br />
<p>We experimentally tested the switch by monitoring production of two fluorescent protein reporters.</p><br />
<p>We confirmed a <b>clear bimodal distribution of reporter fluorescence</b> and demonstrated adoption of <b>stable states</b> <br />
by induction with corresponding inducer molecules.</p><br />
<p>The switch <b>persisted in a stable state</b> after the removal of inducer molecules, which confirmed the <br />
epigenitc bistability of our system.</p><br />
</td><br />
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<h2>Bistable genetic switch based on non-cooperative elements</h2><br />
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<p><br />
<br />
<br />
<br />
<br />
Mathematical analysis of genetic switches from the literature indicates that cooperativity, <br />
which introduces a nonlinear response, is required for a functional bistable switch, <br />
consisting of two mutual repressors (Cherry et al. 2000). Macia et al. (2009) and Widder et al. (2009) <br />
proposed that <b>bistability could be introduced by non-cooperative elements, when nonlinearity is introduced</b> if<br />
for example, protein A is able to repress the transcription of protein B and at the same time activate its own<br />
transcription and <i>vice versa</i> (Figure 1). </p><br />
<br />
<p><br />
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<td class="invisible" style="text-align:justify; width:70%;"><b>Figure 1. Macia et al. (2009) and Widder et al. (2009) proposed a mathematical solution for creation of a bistable switch that does not require cooperative binding of transcriptional regulators.</b> According to the proposed model, transcription factor A should activate its own transcription and repress the transcription of gene B and <i>vice versa</i>.<br />
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<p><br />
<br />
We, as molecular biologists, are not aware of any protein acting simultaneously as a repressor and activator,<br />
therefore we were not surprised that, to our knowledge, this type of a bistable switch has not yet been experimentally<br />
implemented. However, we realized that the ability to design <b>TAL repressors and activators directed against the same <br />
binding site could offer a solution to this problem and provide a unique opportunity to construct orthogonal bistable<br />
switches based on noncooperative elements.</b> </p><br />
<br />
<br />
<br />
<br />
<h2>Results</h2><br />
<br />
<h3>Design</h3><br />
<br />
<p>We designed an <b>upgraded mutual repressor switch</b>, where we introduced <b>two additional positive feedback loops</b> (Figure 2),<br />
consisting of two TAL activators targeted against the same binding sites as the pair of mutual TAL repressors.<br />
In other words, <b>rather than having the same protein function as an activator and repressor, we used two proteins, <br />
competing for the same operator</b>. The same binding sequence for the activator and repressor introduced competition for <br />
the binding site and nonlinearity required for the bistability. For the purpose of our project, <br />
<b>we designed a bistable switch with a positive feedback loop, capable of switching between the two states through regulation<br />
by inducer molecules</b> (Figure 3).<p><br />
<br />
<p><br />
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<b>Figure 2. Scheme of the genetic circuit for the bistable toggle switch composed of a pair of mutual repressors and a pair of autoactivators under the control of two operators.</b><br />
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<b>Figure 3. </b> <b>Designed states of the bistable toogle switch with a positive feedback loop.</b> (A) Addition of inducer 1 triggers dissociation of its cognate repressor from its DNA-binding site. As a consequence, repressor A and activator B are transcribed. Activator B further activates transcription of repressor A and itself, which forms the positive feedback loop of the switch state one. Meanwhile repressor A inhibits the other state of the switch. (B) When the inducer is removed, the inducible repressor binds back to its DNA-binding site, but since activator B is still present, autoactivation is achieved, resulting in a stable state. (C) and (D) depict the switch switched to state two and the function of its other positive feedback loop. <br />
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<h3>Modeling</h3><br />
<p> Before embarking on to the experimental verification, we performed a <a href="https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch">thorough modeling analysis</a> <br />
of the designed switch.<br />
We incorporated parameters obtained from the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators">repression and activation experimental results</a> into our simulations. <br />
Both deterministic and stochastic simulations demonstrate that the positive feedback loop switch is <b>significantly more<br />
stable than the mutual repressor switch</b>. Most importantly, <b>even without cooperativity,<br />
the system exhibits bistability</b> (Figure 4). This switch is also <b>more robust to leaky expression in opposition to the simple mutual repressor switch</b>.</p><br />
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<b>Figure 4. </b> <b>Robustness of the positive feedback loop switch.</b> The positive feedback loop switch exhibited bistability even in the absence of cooperativity and for the minimal promoter's leaky transcription rate of 10% or constitutive promoter leaky transcription rate of 5%. In comparison, the same leaky transcription rate (with equal values of all other parameters) of 5% caused the mutual repressor switch (without the positive feedback loops) to exhibit no bistability unless high cooperativity (2 or more) was introduced into the model.<br />
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<h3>Construction and experimental testing of the bistability of the switch </h3><br />
<br />
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<p><br />
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<br />
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For experimental implementation of the positive feedback loop toggle switch we introduced the following components into cells (Figure 5):<br />
<ul style="padding-left:30px;"><br />
<li>a pair of <a href="https://2012.igem.org/Team:Slovenia/Parts#TALeffectors">TAL:KRAB repressors</a> (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782004">TALA</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782006">TALB</a>), controlled by the opposite TAL (TALA controls the transcription of TALB and TALB controls the transcription of TALA), exactly as in the mutual repressor switch</li><br />
<li>a pair of <a href="https://2012.igem.org/Team:Slovenia/Parts#TALactivators">TAL:VP16 activators</a> (TALA and TALB), each activating its own transcription and transcription of the opposite TAL repressor </li><br />
<li>two of the constructs were tagged with fluorescent reporter proteins (BFP and mCitrine) via a t2A sequence, which ensured the equimolar production of the fluorescent reporter and TAL regulator</li><br />
<li>Both TAL repressor and activator pairs (A and B), controlled by inducible repressors</li><br />
<li>Constitutively expressed inducible repressor constructs</li><br />
<li>Inducer molecules (pristinamycin and erythromycin)</li><br />
</ul> <br />
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<tr class="inliner"><td class="invisible" style="text-align:justify;"><b>Figure 5. Functional components (operons and inducers) of the positive feedback loop switch.</b><br />
.</td></tr><br />
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<br/><br />
<br />
<br />
<br />
<p>We analyzed the performance of the switch by using two fluorescent proteins with good spectral separation <b>(BFP and mCitrine)</b>, which enabled easy detection and quantification by confocal microscopy and flow cytometry.</p><br />
<br />
<br />
<br />
<p>To analyze the bistability we first used flow cytometry, a technique which allowed us to detemine the number of cells <br />
expressing either one or both of the fluorescent reporter proteins. Although cells were transfected with<br />
the complete switch device including both reporters, the analysis of cells demonstrated <b>clear bimodal<br />
distribution</b> - the majority of the analyzed noninduced cells expressed only one of the two fluorescent<br />
proteins, their selection probably resulting from stochastic events or due to a slight imbalance of the amount of<br />
transfected plasmids (Figure 6A). This bimodal distribution of fluorescence <b>clearly demonstrates the <br />
intrinsic bistabilty of our system</b> in comparison to the <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch">mutual repressor switch</a> (classical toggle) topology, <br />
where a large fraction of cells expressed both <a href="https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch#gangnam">fluorescent reporters</a>. <br />
The addition of either one of the inducers switched the reporter production towards the corresponding fluorescent protein (Figures 6B and 6C).</p><br />
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<b>Figure 6. </b> <b>The bistable switch with a positive feedback loop exhibits a bimodal distribution of fluorescence.</b> (A) Non-induced HEK293 cells transfected with plasmids forming the switch (see Figure 5) transcribe either BFP or mCitrine as determined by flow cytometry. (B) HEK293T with the switch plasmids induced with erythromycin (2 mg/L) express mainly mCitrine and (C) cells induced with pristinamycin (2 mg/L) express BFP. Samples were analysed five days after induction.<br />
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<br />
<p>Confocal microscopy confirmed <b>high expression of the expected and no expression of the opposite fluorescent reporter<br />
protein in both induced states</b> (Figure 7). This means the addition of an inducer shifts cells to a corresponding<br />
state which is <b>preserved even when the inducer is removed</b> (Figure 8). The system remained in a stable state several<br />
days after the removal of the signal, which further confirms the epigenetic bistability of our positive feedback loop switch.</p><br />
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<b>Figure 7. </b> <b>Positive feedback loop switch exhibiting two different states at induction with pristinamycine (PI)<br />
and erythromycine (ER) inducer molecules.</b> HEK293T cells were cotransfected with the following plasmids<br />
: PCMV_mCherry transfection control (20 ng), [A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, <br />
[B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16,<br />
PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). <br />
Pristinamycine and erythromycine were added to final concentration of 2 µg/ml.<br />
Fluorescence was measured 3 days after induction. <br />
</td></tr><br />
</tbody><br />
</table><br />
<!-- end table--><br />
<br/><br />
<!-- figure 3 --><br />
<table class="invisible" style="width:100%;"><br />
<tbody class="invisible"><br />
<tr class="invisible"><br />
<td class="invisible"><br />
<img class="invisible" style="width:100%; height:auto;" src="https://static.igem.org/mediawiki/2012/e/ed/Svn12_positivefeedback_8.png"/><br />
</td><br />
</tr> <br />
</tbody><br />
</table><br />
<table class="invisible" style="width:95%; text-align:left;" style="text-align:justify;"><br />
<tbody class="invisible"><br />
<tr class="normal"><td class="invisible" style="text-align:justify;"><br />
<b>Figure 8. </b> <b>Positive feedback loop switch exhibiting stable states at removal of inducer molecules. </b><br />
HEK293T cells were cotransfected with the following plasmids: PCMV_mCherry (20 ng), [A]_PMIN_TALB:KRAB, <br />
[A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB,<br />
PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng).<br />
Pristinamycine and erythromycine were added to final concentration of 2 µg/ml.<br />
Media was replaced 3 days after induction and fluorescence was measured 3 days after removal of inducers.<br />
</td></tr><br />
</tbody><br />
</table><br />
<!-- end table--><br />
<br />
<br /><br />
<h2 style="color:grey;">References</h2><br />
<p style="color:grey;"><br />
Cherry, J., L., Adler, F., R. (2000) How to make a Biological Switch. <i>J. Theor. Biol.</i> <b>203</b>, 117-133.<br />
<br/><br/><br />
Macía, J., Widder, S., Solé, R. (2009) Why are cellular switches Boolean? General conditions for multistable genetic circuits. <i>J. Theor. Biol.</i> <b>261</b>, 126-135. <br />
<br/><br/><br />
Widder, S., Macía, J., and Solé, R. (2009) Monomeric Bistability and the Role of Autoloops in Gene Regulation. <i>PLoS ONE</i> <b>4</b>, e5399.<br />
</p><br />
<br />
<br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'>Controls >></a><br />
</b><br />
<br />
<br />
</div><br />
<!--</div>--><br />
</body><br />
<br />
<br />
</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchControls
Team:Slovenia/TheSwitchControls
2012-10-26T02:55:09Z
<p>Dusanv: </p>
<hr />
<div><html><br />
<head><br />
<meta http-equiv="X-UA-Compatible" content="IE=edge" /><br />
<br />
<style type="text/css"><br />
<br />
<br />
#container {background:#fff; margin:0 auto 0px; padding:5px 0px 0px; width:960px; border-radius:10px;}<br />
#main {background:#fff; width:950px; padding-left:15px; padding-right:15px; padding-top:0px; padding-bottom:10px; margin-top:0px; border-top-left-radius:0px; border-top-right-radius:0px; border-bottom-left-radius:10px; border-bottom-right-radius:10px; font-family: Arial, "Helvetica Neue", Helvetica, sans-serif; font-size:110%; }<br />
<br />
#mainmenu {background:#fff; width:980px; padding-left:0px; padding-right:0px; padding-top:0px; padding-bottom:0px; margin-top:0px; border-top-left-radius:2px; border-top-right-radius:2px; border-bottom-left-radius:10px; border-bottom-right-radius:10px; font-family: Arial, "Helvetica Neue", Helvetica, sans-serif; font-size:110%; }<br />
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<br />
p {line-height:1.5em; margin:0 0 15px; text-align:justify;}<br />
h2 {font-size:1.8em; font-weight:400; margin:0 0 12px;}<br />
<br />
.clearfix:after{clear:both;content:".";display:block;height:0;visibility:hidden;}<br />
* html .clearfix{height:1%;}<br />
*:first-child+html .clearfix{min-height:1%;}<br />
<br />
img { display: block; margin-left: auto; margin-right: auto }<br />
<br />
a, a:visited, a:active {border-bottom:1px dotted #505050; color:#0C5DA5; text-decoration:none;}<br />
a:hover {border-bottom:1px solid #505050; color:#303030; text-decoration:none;}<br />
a img {border:0;}<br />
<br />
.firstHeading {display:none;}<br />
<br />
/*#footer-box { display:none ;}*/<br />
#footer-box { width: 950px; border:none; border-radius:10px; padding:15px 15px 15px 15px; margin-bottom:10px;}<br />
<br />
#top-section {<br />
position: relative;<br />
height: 0px;<br />
width: 965px;<br />
margin: 0 auto 0 auto;<br />
border-left: 1px solid #444444;<br />
border-right: 1px solid #444444;<br />
border-bottom: 0px;<br />
}<br />
<br />
<br />
#catlinks { display:none; }<br />
<br />
<br />
#p-logo { display:none;}<br />
<br />
<br />
#column-one { padding-top:0px; padding: 0 0 0 0;}<br />
<br />
body <br />
{<br />
margin:0; <br />
padding:15px 0 0;<br />
background-color: #ececec;<br />
background-image:url("https://static.igem.org/mediawiki/2012/5/5d/Svn12_bckg_css.png");<br />
background-repeat:repeat;<br />
}<br />
<br />
<br />
#content{<br />
background-color: transparent;<br />
border-left: none;<br />
border-right: none;<br />
width:980px;<br />
}<br />
<br />
#search-controls {<br />
display:none;<br />
}<br />
<br />
<br />
#banner{<br />
background-image:url("https://static.igem.org/mediawiki/2012/a/a9/Svn12_bannertop.png");<br />
background-position: center; <br />
background-repeat: no-repeat;<br />
width:980;<br />
height:239px;<br />
font-family:Arial;<br />
font-size:12px;<br />
padding-left:268px;<br />
}<br />
<br />
<br />
<br />
/* menu (page, edit ...) */<br />
#menubar {<br />
position: absolute;<br />
white-space: nowrap;<br />
top: -6px;<br />
width: 400px;<br />
z-index: 5;<br />
font-family: sans-serif;<br />
font-size: 95%;<br />
line-height: 1em;<br />
}<br />
<br />
.left-menu, .left-menu a {<br />
left: 0px;<br />
text-align: left;<br />
color:#114472;<br />
text-transform: lowercase;<br />
}<br />
<br />
.left-menu:hover {<br />
color: #114472;<br />
background-color: transparent;<br />
}<br />
.right-menu, .right-menu a {<br />
right: 0px;<br />
text-align: right;<br />
color: #114472;<br />
}<br />
#menubar ul {<br />
color: #114472;<br />
list-style: none;<br />
}<br />
#menubar li {<br />
display: inline;<br />
position: relative;<br />
cursor: pointer;<br />
padding-left: 0px;<br />
padding-right: 0px;<br />
}<br />
.left-menu li a {<br />
padding: 0px 10px 0px 0px;<br />
}<br />
.left-menu .selected {<br />
# color: #114472;<br />
}<br />
#.left-menu .selected:hover {<br />
# color: #114472;<br />
#}<br />
<br />
.left-menu:hover a {<br />
color: #114472;;<br />
}<br />
.right-menu li {<br />
# background-color: transparent;<br />
}<br />
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padding: 0px 15px 0px 0px;<br />
color: #114472;;<br />
background-color: transparent;<br />
}<br />
.right-menu li a:hover {<br />
color: #114472;<br />
text-decoration: underline;<br />
}<br />
/* end menu (edit, page ...) */<br />
<br />
<br />
/* Tables */<br />
/* IE 6: http://webdesign.about.com/od/tables/qt/tiptablecenter.htm */<br />
table.normal { border-collapse: collapse; margin: auto; width:70%; margin-bottom:15px; }<br />
td.normal, th.normal { padding-left: 1.4em; padding-right: 1.4em; padding-top: 0.4em; padding-bottom: 0.4em; border: 1px #d8d8d8 solid; }<br />
thead.normal{ background: #0C5DA5; color:#ffffff; border: 1px #d8d8d8 solid; }<br />
tbody .normal{ background: #fff; }<br />
<br />
table.outcome { border-collapse: collapse; margin: auto; width:90%; margin-bottom:15px; }<br />
td.outcome, th.normal { padding-left: 1.4em; padding-right: 1.4em; padding-top: 0.4em; padding-bottom: 0.4em; border: 1px #d8d8d8 solid; }<br />
thead.outcome{ background: #0C5DA5; color:#ffffff; border: 1px #d8d8d8 solid; }<br />
tbody .outcome{ background: #fff; }<br />
ul.circle {<br />
list-style-type:circle;<br />
}<br />
<br />
<br />
/* invisible table */<br />
table.invisible{ border-collapse: collapse; margin: auto; width:100%; margin-bottom:15px; }<br />
td.invisible, th.invisible { padding-left: 1.4em; padding-right: 1.4em; padding-top: 0.4em; padding-bottom: 0.4em; border:none; }<br />
thead.invisible{ background: #0C5DA5; color:#ffffff; border:none; }<br />
tbody .invisible{ background: #fff; }<br />
<br />
<br />
/* summary table */<br />
table.summary{ border-collapse: collapse; margin: auto; width:100%; margin-bottom:10px; }<br />
td.summary, th.summary{ background:#d0ecf4; color:#000000; padding-left: 5px; padding-right: 15px; padding-top: 15px; padding-bottom: 15px; border:none; border-radius:0px;}<br />
thead.summary{ background:#d0ecf4; color:#000000; border:none; border-radius:0px; }<br />
tbody.summary{ background: #fff; }<br />
<br />
<br />
<br />
/* CSS navigation menu (blue) */<br />
#cssmenu{ height:37px; display:block; padding:0; margin:0; border:1px solid; border-radius:0px; } <br />
#cssmenu > ul {list-style:inside none; padding:0; margin:0;} <br />
#cssmenu > ul > li {list-style:inside none; padding:0; margin:0; float:left; display:block; position:relative;} <br />
#cssmenu > ul > li > a{ outline:none; display:block; position:relative; padding:12px 17px; font:bold 13px/100% Arial, Helvetica, sans-serif; text-align:center; text-decoration:none; text-shadow:1px 1px 0 rgba(0,0,0, 0.4); } <br />
#cssmenu > ul > li:first-child > a{border-radius:5px 0 0 5px;} <br />
#cssmenu > ul > li > a:after{ content:''; position:absolute; border-right:1px solid; top:-1px; bottom:-1px; right:-2px; z-index:99; } <br />
#cssmenu ul li.has-sub:hover > a:after{top:0; bottom:0;} <br />
#cssmenu > ul > li.has-sub > a:before{ content:''; position:absolute; top:18px; right:6px; border:5px solid transparent; border-top:5px solid #fff; } <br />
#cssmenu > ul > li.has-sub:hover > a:before{top:19px;} <br />
#cssmenu ul li.has-sub:hover > a{ background:#043A6B; border-color:#3f3f3f; padding-bottom:13px; padding-top:13px; top:-1px; z-index:999; } <br />
#cssmenu ul li.has-sub:hover > ul, #cssmenu ul li.has-sub:hover > div{display:block;} <br />
#cssmenu ul li.has-sub > a:hover{background:#043A6B; border-color:#3f3f3f;} <br />
#cssmenu ul li > ul, #cssmenu ul li > div{ display:none; width:auto; position:absolute; top:38px; padding:10px 0; background:#043A6B; border-radius:0 5px 5px 5px; z-index:999; } <br />
/*#cssmenu ul li > ul{width:200px;} */<br />
#cssmenu ul li > ul{width:165px;}<br />
#cssmenu ul li > ul li{display:block; list-style:inside none; padding:0; margin:0; position:relative;} <br />
#cssmenu ul li > ul li a{ outline:none; display:block; position:relative; margin:0; padding:8px 20px; font:10pt Arial, Helvetica, sans-serif; color:#fff; text-decoration:none; text-shadow:1px 1px 0 rgba(0,0,0, 0.5); } <br />
#cssmenu ul li > ul li a table tr td span{ outline:none; display:block; position:relative; margin:0; padding:0px 0px; font:10pt Arial, Helvetica, sans-serif; color:#fff; text-decoration:none; text-shadow:1px 1px 0 rgba(0,0,0, 0.5); } <br />
#cssmenu, #cssmenu > ul > li > ul > li a:hover<br />
{ background:#043A6B; <br />
background:-moz-linear-gradient(top, #043A6B 0%, #0C5DA5 100%); <br />
background:-webkit-gradient(linear, left top, left bottom, color-stop(0%,#043A6B), color-stop(100%,#0C5DA5)); <br />
background:-webkit-linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
background:-o-linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
background:-ms-linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
background:linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
filter:progid:DXImageTransform.Microsoft.gradient(startColorstr='#043A6B', endColorstr='#0C5DA5',GradientType=0); <br />
} <br />
#cssmenu{border-color:#043A6B;} <br />
#cssmenu > ul > li > a{border-right:1px solid #043A6B; color:#fff;} <br />
#cssmenu > ul > li > a:after{border-color:#0C5DA5;} <br />
#cssmenu > ul > li > a:hover{background:#0C5DA5;} <br />
/* end CSS navigation menu (blue) */<br />
<br />
<br />
ul {<br />
line-height: 1.5em;<br />
list-style-type: square;<br />
margin: 0 0 0 0;<br />
padding: 0;<br />
/*list-style-image: url(bullet.gif);*/<br />
}<br />
<br />
.abstract {background:#0C5DA5; color:#ffffff; padding:10px; border-radius:5px;}<br />
<br />
<br />
<br />
table.splash {}<br />
td.splash, th.splash {}<br />
thead.splash{ background: #0C5DA5; color:#ffffff; border: 0px #d8d8d8 solid; }<br />
tbody .splash{ background: #fff; }<br />
<br />
table.newtable {background-color:transparent;}<br />
td.newtable, th.newtable {background-color:transparent;}<br />
thead.newtable{ }<br />
tbody .newtable{}<br />
<br />
<br />
td.splash .ttip{<br />
float:left;<br />
position:absolute;<br />
left:150px;<br />
top:660px;<br />
visibility:hidden;<br />
z-index:100;<br />
width:700px;<br />
opacity:0.85;<br />
background:#dedede;<br />
border-radius: 30px;<br />
font-family: Arial, "Helvetica Neue", Helvetica, sans-serif;<br />
padding: 10px 10px 10px 10px;<br />
}<br />
<br />
td.splash .ttip2{<br />
float:left;<br />
position:absolute;<br />
left:150px;<br />
top:960px;<br />
visibility:hidden;<br />
z-index:100;<br />
width:700px;<br />
opacity:0.85;<br />
background:#dedede;<br />
border-radius: 30px;<br />
font-family: Arial, "Helvetica Neue", Helvetica, sans-serif;<br />
padding: 10px 10px 10px 10px;<br />
}<br />
<br />
td.splash:hover > .ttip<br />
{<br />
visibility:visible;<br />
} <br />
<br />
td.splash:hover > .ttip2<br />
{<br />
visibility:visible;<br />
} <br />
<br />
/*<br />
td.splash .pic{<br />
position:absolute;<br />
visibility:visible;<br />
z-index:1;<br />
}<br />
<br />
td.splash .pic2{<br />
position:absolute;<br />
visibility:hidden;<br />
z-index:10;<br />
}<br />
<br />
td.splash:hover > .ttip<br />
{<br />
visibility:visible;<br />
} <br />
<br />
<br />
td.splash:hover .pic<br />
{<br />
<br />
} <br />
*/<br />
<br />
</style><br />
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<!--<div id="container">--><br />
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<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<!-- end main menu --><br />
<br />
</div> <!-- end menu --><br />
<br />
<br />
<div id="main"><br />
<br />
<br/><br />
<h2>Controls</h2><br />
<br />
<br />
<br />
<br />
<hr><br />
<b><br />
Next: <a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'>Safety mechanisms >></a><br />
</b><br />
<br />
</div><br />
<!--</div>--><br />
<br />
<br />
<br />
</body><br />
<br />
<br />
</html></div>
Dusanv
http://2012.igem.org/Team:Slovenia/TheSwitchControls
Team:Slovenia/TheSwitchControls
2012-10-26T02:53:54Z
<p>Dusanv: </p>
<hr />
<div><html><br />
<head><br />
<meta http-equiv="X-UA-Compatible" content="IE=edge" /><br />
<br />
<style type="text/css"><br />
<br />
<br />
#container {background:#fff; margin:0 auto 0px; padding:5px 0px 0px; width:960px; border-radius:10px;}<br />
#main {background:#fff; width:950px; padding-left:15px; padding-right:15px; padding-top:0px; padding-bottom:10px; margin-top:0px; border-top-left-radius:0px; border-top-right-radius:0px; border-bottom-left-radius:10px; border-bottom-right-radius:10px; font-family: Arial, "Helvetica Neue", Helvetica, sans-serif; font-size:110%; }<br />
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<br />
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<br />
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.left-menu, .left-menu a {<br />
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right: 0px;<br />
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color: #114472;<br />
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#menubar ul {<br />
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#menubar li {<br />
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position: relative;<br />
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padding-left: 0px;<br />
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.left-menu li a {<br />
padding: 0px 10px 0px 0px;<br />
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thead.outcome{ background: #0C5DA5; color:#ffffff; border: 1px #d8d8d8 solid; }<br />
tbody .outcome{ background: #fff; }<br />
ul.circle {<br />
list-style-type:circle;<br />
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table.summary{ border-collapse: collapse; margin: auto; width:100%; margin-bottom:10px; }<br />
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/* CSS navigation menu (blue) */<br />
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#cssmenu > ul > li {list-style:inside none; padding:0; margin:0; float:left; display:block; position:relative;} <br />
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#cssmenu > ul > li:first-child > a{border-radius:5px 0 0 5px;} <br />
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#cssmenu > ul > li.has-sub > a:before{ content:''; position:absolute; top:18px; right:6px; border:5px solid transparent; border-top:5px solid #fff; } <br />
#cssmenu > ul > li.has-sub:hover > a:before{top:19px;} <br />
#cssmenu ul li.has-sub:hover > a{ background:#043A6B; border-color:#3f3f3f; padding-bottom:13px; padding-top:13px; top:-1px; z-index:999; } <br />
#cssmenu ul li.has-sub:hover > ul, #cssmenu ul li.has-sub:hover > div{display:block;} <br />
#cssmenu ul li.has-sub > a:hover{background:#043A6B; border-color:#3f3f3f;} <br />
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/*#cssmenu ul li > ul{width:200px;} */<br />
#cssmenu ul li > ul{width:165px;}<br />
#cssmenu ul li > ul li{display:block; list-style:inside none; padding:0; margin:0; position:relative;} <br />
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#cssmenu, #cssmenu > ul > li > ul > li a:hover<br />
{ background:#043A6B; <br />
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background:-webkit-linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
background:-o-linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
background:-ms-linear-gradient(top, #043A6B 0%,#0C5DA5 100%); <br />
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#cssmenu{border-color:#043A6B;} <br />
#cssmenu > ul > li > a{border-right:1px solid #043A6B; color:#fff;} <br />
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#cssmenu > ul > li > a:hover{background:#0C5DA5;} <br />
/* end CSS navigation menu (blue) */<br />
<br />
<br />
ul {<br />
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<br />
.abstract {background:#0C5DA5; color:#ffffff; padding:10px; border-radius:5px;}<br />
<br />
<br />
<br />
table.splash {}<br />
td.splash, th.splash {}<br />
thead.splash{ background: #0C5DA5; color:#ffffff; border: 0px #d8d8d8 solid; }<br />
tbody .splash{ background: #fff; }<br />
<br />
table.newtable {background-color:transparent;}<br />
td.newtable, th.newtable {background-color:transparent;}<br />
thead.newtable{ }<br />
tbody .newtable{}<br />
<br />
<br />
td.splash .ttip{<br />
float:left;<br />
position:absolute;<br />
left:150px;<br />
top:660px;<br />
visibility:hidden;<br />
z-index:100;<br />
width:700px;<br />
opacity:0.85;<br />
background:#dedede;<br />
border-radius: 30px;<br />
font-family: Arial, "Helvetica Neue", Helvetica, sans-serif;<br />
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}<br />
<br />
td.splash .ttip2{<br />
float:left;<br />
position:absolute;<br />
left:150px;<br />
top:960px;<br />
visibility:hidden;<br />
z-index:100;<br />
width:700px;<br />
opacity:0.85;<br />
background:#dedede;<br />
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{<br />
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<br />
td.splash:hover > .ttip2<br />
{<br />
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} <br />
<br />
/*<br />
td.splash .pic{<br />
position:absolute;<br />
visibility:visible;<br />
z-index:1;<br />
}<br />
<br />
td.splash .pic2{<br />
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z-index:10;<br />
}<br />
<br />
td.splash:hover > .ttip<br />
{<br />
visibility:visible;<br />
} <br />
<br />
<br />
td.splash:hover .pic<br />
{<br />
<br />
} <br />
*/<br />
<br />
</style><br />
<br />
<br />
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</head><br />
<br />
<body><br />
<div id="banner"><br />
<a style="position:absolute; top:0px; left:490px;" href="https://2012.igem.org/Main_Page"><b></b></a><br />
</div><br />
<br />
<br />
<!--<div id="container">--><br />
<div id="mainmenu"><br />
<br />
<!-- start main menu --><br />
<div id='cssmenu'><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Idea</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaBioTherapy'><span>Biological therapy</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/IdeaChallenge'><span>Challenge</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Idea'><span>Solution</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>The switch</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitch'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Safety mechanisms</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
</ul><br />
</li><br />
<br />
<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li><br />
<br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> <br />
</ul><br />
</li><br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li> <br />
</ul><br />
</li><br />
<br />
<br />
<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a><br />
<ul><br />
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li><br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> <br />
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li> <br />
</ul><br />
</li><br />
</ul><br />
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