Team:Imperial College London/Temporal Control

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=[[Image:II09_Temporal_control.png|50px]]Temporal Control=
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=[[Image:II09_Temporal_control.png|50px]]<font face='Calibri' size='5'><b>Module Integration</b></font>=
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[[Image:II09_Timeline.png|600px|centre]]
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The figure above depicts temporal control in relation to [https://2009.igem.org/Team:Imperial_College_London/M1 Module 1], [https://2009.igem.org/Team:Imperial_College_London/M2 Module 2], and [https://2009.igem.org/Team:Imperial_College_London/M3 Module 3] with expected changes in OD, carbon source concentration, RFP and GFP <b>(LINK TO ASSAYS/TESTING CONSTRUCTS)</B>.
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In <i>The E.ncapsulator</i>, temporal control is an integral part of the design. The 3 modules can only perform their function successfully in the system if they have a temporal mechanism triggering them in a sequential manner. We have achieved this using 3 types of control:
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<html>
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*<b>Chemical induction</b> starts protein drug production.
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<div class="highslide-gallery" align="center">
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*<b>Autoinduction</b> starts encapsulation.
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<a href="https://static.igem.org/mediawiki/2009/2/29/Ii09_tcg1.PNG" class="highslide" onclick="return hs.expand(this)">
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*<b>Thermoinduction</b> starts genomic deletion.
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<img src="https://static.igem.org/mediawiki/2009/2/29/Ii09_tcg1.PNG" alt="" title="Testing Click to expand" width="100%"/>
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</a>
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<div class="highslide-caption">
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Temporal Control
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<br>
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<br>
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<font face='Calibri' size='4'><b>Engineering Approach to Module Integration by <u>Temporal Control</u></b></font>
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== Chemical induction ==
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This <b>temporal control</b> platform showcases our engineering approach in the E.ncapsulator project. It allows us to integrate all our modules in a simple and elegant way.
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<!--[WHAT IS IPTG? HOW DOES IT ACT?]-->
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From the [https://2009.igem.org/Team:Imperial_College_London/M1/Genetic Module 1 genetic circuit], in the absence of IPTG in the system, the LacI repressor inhibits production of the protein of interest.  
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When IPTG is added in we start the production of the drug of interest:
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[[Image:II09_NoIPTG_yesIPTG.jpg|400px|centre]]
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<!--[For more details of the system, click here- (bits of Charles' literature review), includes info on lac operon and how we have modified it]-->
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We’ve made our entire system <b>modular</b>, where we trigger the succession of events using this control system. Here, we have tackled a drug delivery problem, but this "Black box" approach can be applied to any other system. This is our <b>novel engineering approach</b>, which is extremely <b>reusable</b> in synthetic biology.
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== Autoinduction ==
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[[Image:II09_BlackBox3.png|400px|center]]<br>
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The CRP glucose repressible promoter <!--add in a link of some sort--> triggers the encapuslation phase.
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*When levels of <b>glucose</b> in the medium are <b>high</b>, <b>cAMP</b> levels are <b>low</b> and encapsulation is <b>repressed</b>
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<!--<html>
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*When the levels of <b>glucose</b> in the medium are <b>low</b>, <b>cAMP</b> levels are <b>high</b> and encapsulation is <b>induced</b>.
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<div class="highslide-gallery" align="center">
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[[Image:II09_encapsulate_noencapsulate.jpg|400px|centre]]
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<a href="https://static.igem.org/mediawiki/2009/3/3c/Ii09_tempotxt.PNG" class="highslide" onclick="return hs.expand(this)">
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Starting the encapsulation and maintaining expression of colanic acid requires energy in the form of carbon sources. Bacteria use up a primary source to grow and trigger encapsulation. Glucose here is the primary source, which in unison with cAMP, is responsible for switching on the CRP promoter and the encapsulation process.A secondary carbon source is also needed to maintain the system once glucose is used up. This phenomenon of switching from a primary source to a secondary source is known as a diauxie.  
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<img src="https://static.igem.org/mediawiki/2009/3/3c/Ii09_tempotxt.PNG" alt="" title="Testing Click to expand" width="100%"/>
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[[Image:II09_diauxi_illust.jpg|300px|centre]]
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</a>
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This switching process occurs automatically, hence we have called it "autoinduction".
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<div class="highslide-caption">
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Temporal Control
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</div>
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</div>
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</html>-->
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<!--====Testing Construct====
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This testing construct was used to test the inducible promoters using flourescent proteins as output reporters.<br>
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<html>
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<div class="highslide-gallery" align="center">
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<a href="https://static.igem.org/mediawiki/2009/0/0c/II09_Temp_Construct.png" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/0/0c/II09_Temp_Construct.png" alt="" title="Testing Construct used for Temporal Control Timeline" width="50%"/>
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<div class="highslide-caption">
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Key to genetic circuit diagrams
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</div>
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-->
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===Timeline of Temporal Control===
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This timeline shows the sequence of occurrence of these events:
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/1/15/II09_Timeline.png" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/1/15/II09_Timeline.png" alt="" title="Module 3 is the destruction of the genetic material." width="95%"/>
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<a href="https://static.igem.org/mediawiki/2009/3/3d/Ii09_tcg4.PNG" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/3/3d/Ii09_tcg4.PNG" alt="" title="Module 3 is the destruction of the genetic material." width="95%"/>
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<a href="https://static.igem.org/mediawiki/2009/7/7f/Ii09_tcg7.PNG" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/7/7f/Ii09_tcg7.PNG" alt="" title="Module 3 is the destruction of the genetic material." width="95%"/>
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<a href="https://static.igem.org/mediawiki/2009/8/80/Ii09_tcg8.PNG" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/8/80/Ii09_tcg8.PNG" alt="" title="Module 3 is the destruction of the genetic material." width="95%"/>
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</a>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Graph#Explanation_of_Timeline
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"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; <b>About the timeline, its explainations, and to view our testing construct.</b>
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<!--In our project there are 3 forms of temporal control that have been implemented.
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* <b>Chemical induction</b>: Triggers the production of the polypeptide of interest using IPTG. Effectively 'kicks off' the system once the cell density is high enough.
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* <b>Autoinduction</b>: Represses encapsulation when glucose levels are high, and kick starts it once glucose is used up. This allows a sufficient amount of protein production to have taken place before the cell focuses its resources on encapsulation.
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* <b>Thermoinduction</b>: Triggers genome deletion when the temperature is increased. Thermoinduction was necessary, as chemical induction may be blocked by the presence of the capsule (that inhibits diffusion).<br><br><br>-->
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<!--===Testing Construct===
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This testing construct was used to test the inducible promoters using flourescent proteins as output reporters.<br>
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<html>
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<div class="highslide-gallery" align="center">
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<a href="https://static.igem.org/mediawiki/2009/0/0c/II09_Temp_Construct.png" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/0/0c/II09_Temp_Construct.png" alt="" title="Testing Construct used for Temporal Control Timeline" width="50%"/>
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</a>
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<div class="highslide-caption">
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Key to genetic circuit diagrams
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</div>
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</div>
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</html>
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The timeline shows the sequence of occurrence of these events:
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<html>
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<center>
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/1/15/II09_Timeline.png" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/1/15/II09_Timeline.png" alt="" title="Module 3 is the destruction of the genetic material." width="95%"/>
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</a>
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</div>
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</center>
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</html>
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[[Team:Imperial_College_London/Temporal_Control/Graph#Testing_Construct | Click here]] to see our testing construct.
<!--
<!--
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[Diauxic growth and autoinduction media] CRP promoter
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===Explanation of Timeline===
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<b>Carbon Source Concentration</b>:
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*Glucose
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The primary carbon source is glucose, as this is preferentially used by the cell.
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*Secondary carbon source
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This is used by the cell after glucose in the media is depleted.
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By carefully balancing the initial concentrations of these carbon sources, The E.ncapsulator will begin Encapsulation (Module 2) only once protein production is at sufficiently high levels.
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<b>OD 600:</b>
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OD600 corresponds to the optical density of the cells. At sufficiently low levels of cell density, the absorbance of light of wavelength 600nm has a linear relationship with the cell density. This plot therefore models the growth of the cells throughout.
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<b>RFP / OD</b>:
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RFP is a red coloured flourescent protein that is commonly used as a reporter. The gene coding for this protein is part of the same operon as the protein of interest. As the protein of interest is produced in Module 1, RFP is coexpressed alongside. The RFP must be normalised against optical density (shown above), as the cell density is increasing throughout.
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<b>GFP / OD </b>:
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GFP is a green coloured flourescent protein that is also commonly used as a reporter. The coding gene is under control of the same promoter as the genes for <b>Module 2</b>. This means that GFP expression is tied into encapsulation. The GFP again must be normalised against optical density (shown above) to account for the increase in cell density.
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<br>
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<br>
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<!--===Explainations for temporal overview===
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<i><b> Please click on the table for a clearer view. </b></i>
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<html>
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<center>
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/5/55/Ii09_temporalctrloverviewtxt.png" class="highslide" onclick="return hs.expand(this)">
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<img src="https://static.igem.org/mediawiki/2009/5/55/Ii09_temporalctrloverviewtxt.png" alt="" title="Temporal induction overview" width="95%"/>
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</a>
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</div>
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</center>
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</html>
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<!--This timeline shows the sequence of events occuring within the system. <br>
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*Starting from the top, we have the different carbon sources responsible for autoinduction of the system. The primary carbon source is glucose, as this is preferentially used by the cell. Also present in the media is a secondary carbon source, which is used by the cell after glucose in the media is depleted. By carefully balancing the initial concentrations of these carbon sources, <b><i>The E.ncapsulator</i></b> will begin <b>Module 2</b> only once protein production is at sufficiently high levels.
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*OD600 corresponds to the optical density of the cells. At sufficiently low levels of cell density, the absorbance of light of wavelength 600nm has a linear relationship with the cell density. This plot therefore models the growth of the cells throughout.
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*RFP is a red coloured flourescent protein that is commonly used as a reporter. The gene coding for this protein is part of the same operon as the protein of interest. As the protein of interest is produced in Module 1, RFP is coexpressed alongside. The gene for RFP is under the control of the chemically induced promoter, and we can see an increase in flourescence when induced with IPTG. The RFP must be normalised against optical density (shown above), as the cell density is increasing throughout.
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*[http://en.wikipedia.org/wiki/Green_fluorescent_protein GFP] is  a green coloured flourescent protein that is also commonly used as a reporter. The coding gene is under control of the same promoter as the genes for Module 2. This means that GFP expression is tied into the module, and we can see the rise in GFP levels that correlate with the switch to the secondary carbon source and therefore the start of Module 2. The GFP again must be normalised against optical density (shown above) to account for the increase in cell density.-->
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<br>
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<!--===Our Results===
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<u>Wetlab</u>
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//graph to be decided
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Autoinduction
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"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; About the wetlab results!
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<br>
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<u>Drylab</u>
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[[Image:II09_diax_wolf.jpg | left]]We have modelled autoinduction by a number of models, and we hope that experimental results will confirm which model is most applicable to our system.
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The first of our diauxic growth models is a cybernetic model developed by Kompala et al [1]. This model shows that glucose (S1) is used up before the secondary carbon source (S2). During this phase, the population (X) is in exponential grow until glucose (S1) runs out. This is followed by a stationary growth phase, and finally the population enters a second exponential growth phase as they start uptaking the secondary carbon source (S2).
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Autoinduction
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"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; about the models and simulations!
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<br> <br>
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====References====
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[1]Kompala, D.S., D. Ramkrishna and G.T. Tsao, "Cybernetic modeling of microbial growth on multiple substrates," Biotechnology and Bioengineering 26 :1272-1281 (1984).
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<center>
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-->
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==Conclusion==
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Module integration has allowed us to link all our modules together, to create a working network.
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It also allows us to showcase the advantages of <b>synthetic biology</b> as the <b>marriage of engineering with biology</b>. By integrating the engineering principles of reusability and simplicity of system, we have developed this platform to program biologcal systems.
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<br>
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<br>
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<center>
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===Project Tour===
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/Module3L.jpg"></a>
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<a href="https://2009.igem.org/Team:Imperial_College_London/Genetic_Circuit"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/GeneticCircuitsR.jpg"></a>
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</html>
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<hr>
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===Temporal Control Contents===
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Chemical_Induction"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_chemicalinduction.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Autoinduction"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Thermoinduction"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Thermoinduction1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Temporal_Control"><img style="vertical-align:bottom;"width="20%"src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Autoinduction"><img style="vertical-align:bottom;" width="20%"  src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage6.png"></a></center></html>
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[click for more details... link to models too]-->
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<html><table border="0" style="background-color:transparent;" width="100%">
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<tr><td width="0%"></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Chemical_Induction"><b>Chemoinduction</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Autoinduction"><b>Autoinduction</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Thermoinduction"><b>Thermoinduction</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Temporal_Control"><b>Wet Lab</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Autoinduction"><b>Modelling</b></a></center></td>
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<tr><td width="1%"></td>
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== Thermoinduction ==
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<td width="1%"></td>
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This part of temporal control is responsible for triggering genomic deletion, after encapsulation has taken place.
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</tr></table></html>
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*When the <b>temperature</b> is <b>low</b>, restriction enzymes are inhibited and <b>DNA is protected</b>.
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*When the <b>temperature</b> is <b>high</b>, restriction enzymes are produced and <b>DNA is digested</b>.
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[[Image:II09_hitemp_lotemp.jpg|400px|centre]]
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{{Imperial/09/TemplateBottom}}
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Latest revision as of 01:00, 21 October 2009

Contents

II09 Temporal control.pngModule Integration

Temporal Control


Engineering Approach to Module Integration by Temporal Control

This temporal control platform showcases our engineering approach in the E.ncapsulator project. It allows us to integrate all our modules in a simple and elegant way.

We’ve made our entire system modular, where we trigger the succession of events using this control system. Here, we have tackled a drug delivery problem, but this "Black box" approach can be applied to any other system. This is our novel engineering approach, which is extremely reusable in synthetic biology.

II09 BlackBox3.png


Timeline of Temporal Control

This timeline shows the sequence of occurrence of these events:



  About the timeline, its explainations, and to view our testing construct.




Conclusion

Module integration has allowed us to link all our modules together, to create a working network.

It also allows us to showcase the advantages of synthetic biology as the marriage of engineering with biology. By integrating the engineering principles of reusability and simplicity of system, we have developed this platform to program biologcal systems.

Project Tour

Temporal Control Contents

Chemoinduction
Autoinduction
Thermoinduction
Wet Lab
Modelling

Mr. Gene   Geneart   Clontech   Giant Microbes
Retrieved from "http://2009.igem.org/Team:Imperial_College_London/Temporal_Control"