Team:Michigan/Project

From 2008.igem.org

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= '''<font color=dodgerblue size=6>Project Description</font>''' =
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= '''<font color=royalblue size=6>Project Description</font>''' =
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== <font color=dodgerblue size=4><B>Circadian Clocks</B></font> ==
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== <font color=royalblue size=4>Circadian Clocks</font> ==
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Short background on circadian clocks... why they're important, why they're studied, maybe who studies them...  
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The human body’s circadian rhythm or “clock” regulates the daily cycles of many physiological and metabolic processes such as the sleep wake cycle and feeding rhythms. The biological processes and temporal coordination are crucial to the health and survival of organisms. The processes occur with the capacity to oscillate with a wide variety of periods that are controlled by the interplay of numerous molecular factors that orchestrate complex feedback loops and processes that are fundamentally mediated by gene expression and the events that follow.  
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== <font color=dodgerblue size=4><B>Our Project: The Sequestillator</B></font> ==
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In humans, circadian rhythm arises from circadian complexity and involves the activity of several components, most importantly the negative elements period homologues PER1 PER2 and the CRY1 and CRY2 crytpochrome elements along with the positive acting proteins of CLOCKA and BMAL1.
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The figure to the right depicts the molecular interactions in mammalian circadian feedback loops. The oscillator is composed of interlocking feedback loops that regulate the abundance and activity of transcription factors. These transcription factors control the expression of genes in the output pathways from the oscillator, resulting in behavioral and physiological rhythms.
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The CLOCK and BMAL1 form heterodimers and activate transcription of the Per and Cry genes that form the period. The critical mechanism in the pathway occurs when PER and CRY proteins bind as heterodimers and inhibit CLOCK and BMAL1 transcription via sequestration.
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[[Image:Circadian clock.PNG|450px]]
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== <font color=royalblue size=4>Our Project: The Sequestillator</font> ==
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We can subdivide our clock into two parts:  the activator module and the repressor module.  The activator module consists of the constitutive promoter driving the NifA gene, thereby producing a constant amount of NifA. We will be using three different BioBrick promoters - corresponding to low, medium, and high outputs of NifA respectively.  The NifA protein  binds to the nifHp promoter of the repressor module, activating transcription of the NifL gene.  Once NifL dimerizes, it can bind to the NifA hexamer, hence preventing NifA from binding to NifHp.  This sequestration effect provides the clock's negative feedback loop that is essential for oscillations.
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Our hope is to put these modules on the E.coli chromosome using a BioBrick compatible Arabinose Landing Pad (our iGEM 2007 project) and a Leucine Landing Pad (a construct of a former Ninfa lab member, Dong Eun Chang).  We will use E.coli strain NCM 1971, which has the nifHp driving lacZ on the chromosome.  This way, we can test the amounts of NifA via Beta-galactosidase activity and test the amount of NifL via fluorescent microscopy.
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<div align=center>[[Image:New full topology - gold 2 new.png|500px]]</div>
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<br><div align=center>[[Image:Full topology.png]]</div><br>
 
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We can subdivide our clock into three parts: the tetR system, the activator module, and the repressor module.  The activator module consists of the tet promoter driving the NifA gene.  The NifA protein  binds to the nifHp promoter of the repressor module, activating transcription of the NifL gene.  Once NifL dimerizes, it can bind to the NifA hexamer, hence preventing NifA from binding to NifHp.  This sequestration effect provides the clock's negative feedback loop that is essential for oscillations.
 
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The tetR system regulates the levels of nifA.  A constitutive promoter drives the TetR gene.  At its resting state of our circuit, TetR completely represses the production of NifA.  However, addition of anhydrotetracycline prevents the Tet protein from binding to tet promoter, allowing for a "constitutive" production of NifA as long as the levels of anhydrotetracycline are kept constant.  This module allows us to tune the levels of NifA by adjusting the amount of anhydrotetracycline we add.
 
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= '''<font color=dodgerblue size=6>Sequestilator Modeling</font>''' =
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= '''<font color=royalblue size=6>Sequestillator Modeling</font>''' =
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<div align=center>[[Image:Stochastic.png]]</div>
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<div align=center>[[Image:Stochastic.png]]</div><br><br>
<div align=center>[[Team:Michigan/Project/Modeling]]</div>
<div align=center>[[Team:Michigan/Project/Modeling]]</div>
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= '''<font color=dodgerblue size=6>Sequestilator Fabrication</font>''' =
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If you like the way this looks, you could put a summary of what you built here and then we can have a separate page for fabrication, which might be a good idea.
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= '''<font color=royalblue size=6>Sequestillator Fabrication</font>''' =
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[[Team:Michigan/Project/Fabrication]]
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<br><br><div align=center>[[Image:Fabrication.PNG|400px]]</div><br><br>
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<br><div align=center>[[Team:Michigan/Project/Fabrication]]</div>
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= '''<font color=dodgerblue size=6>Landing Pads</font>''' =
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= '''<font color=royalblue size=6>Landing Pads</font>''' =
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A landing pad is tool used to place synthetic operons on a chromosome.  We will be using two landing pads for our project: the arabinose landing pad and leucine landing pad.  Both of these plasmids will replace the respective metabolic operons with our desired subcloned genetic elements.  The leucine landing pad was constructed by a former member of the Ninfa lab, Dong Eun Chang.  The arabinose landing pad was a part of our iGEM 2007 project, and was worked on by Alyssa Delke and Khalid Miri.  Our reason for using these pieces is to limit the noise in our system so that hopefully we can see more sustained oscillations than previous synthetic clocks have given.
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A landing pad is tool that can be used by all synthetic biologists to insert synthetic operons onto the chromosome of <i>E. coli</i>.  We will be using two landing pads for our project: the arabinose landing pad and leucine landing pad.  Both of these landing pads will replace the respective metabolic operons with our desired subcloned genetic elements.  The leucine landing pad was constructed by a former member of the Ninfa lab, Dong Eun Chang and the arabinose landing pad was a part of our iGEM 2007 project, and was worked on by Alyssa Delke and Khalid Miri.  In using these landing pads, we wish to limit the noise in our system in order to (hopefully) obtain more sustained oscillations than previous synthetic clocks have given.
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<br><div align=center> [[Image:Landing Pad Polylinker.png]]<br>BioBrick Landing Pad Polylinker<br><br>
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<div align=center>[[Team:Michigan/Project/LandingPads]]</div>
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[[Image:Landing pad plasmid.png]]<br>Arabinose Landing Pad with our second operon inserted<br><br>
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[[Image:Landing pad plasmid - gold.png|400px|LP]]
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[http://synthbio.engin.umich.edu/wiki/index.php/BioBrick_Landing_Pad <font color=navy size=3> Learn more about Landing Pads Here!</font>]
 
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Latest revision as of 02:09, 30 October 2008


Michigan iGEM website header.jpg

HOME THE TEAM THE PROJECT REGISTRY PARTS NOTEBOOK


Project Description

Circadian Clocks

The human body’s circadian rhythm or “clock” regulates the daily cycles of many physiological and metabolic processes such as the sleep wake cycle and feeding rhythms. The biological processes and temporal coordination are crucial to the health and survival of organisms. The processes occur with the capacity to oscillate with a wide variety of periods that are controlled by the interplay of numerous molecular factors that orchestrate complex feedback loops and processes that are fundamentally mediated by gene expression and the events that follow.

In humans, circadian rhythm arises from circadian complexity and involves the activity of several components, most importantly the negative elements period homologues PER1 PER2 and the CRY1 and CRY2 crytpochrome elements along with the positive acting proteins of CLOCKA and BMAL1.

The figure to the right depicts the molecular interactions in mammalian circadian feedback loops. The oscillator is composed of interlocking feedback loops that regulate the abundance and activity of transcription factors. These transcription factors control the expression of genes in the output pathways from the oscillator, resulting in behavioral and physiological rhythms.

The CLOCK and BMAL1 form heterodimers and activate transcription of the Per and Cry genes that form the period. The critical mechanism in the pathway occurs when PER and CRY proteins bind as heterodimers and inhibit CLOCK and BMAL1 transcription via sequestration.

Circadian clock.PNG

Our Project: The Sequestillator

We can subdivide our clock into two parts: the activator module and the repressor module. The activator module consists of the constitutive promoter driving the NifA gene, thereby producing a constant amount of NifA. We will be using three different BioBrick promoters - corresponding to low, medium, and high outputs of NifA respectively. The NifA protein binds to the nifHp promoter of the repressor module, activating transcription of the NifL gene. Once NifL dimerizes, it can bind to the NifA hexamer, hence preventing NifA from binding to NifHp. This sequestration effect provides the clock's negative feedback loop that is essential for oscillations. Our hope is to put these modules on the E.coli chromosome using a BioBrick compatible Arabinose Landing Pad (our iGEM 2007 project) and a Leucine Landing Pad (a construct of a former Ninfa lab member, Dong Eun Chang). We will use E.coli strain NCM 1971, which has the nifHp driving lacZ on the chromosome. This way, we can test the amounts of NifA via Beta-galactosidase activity and test the amount of NifL via fluorescent microscopy.

New full topology - gold 2 new.png



Sequestillator Modeling

Stochastic.png


Sequestillator Fabrication



Fabrication.PNG




Landing Pads

A landing pad is tool that can be used by all synthetic biologists to insert synthetic operons onto the chromosome of E. coli. We will be using two landing pads for our project: the arabinose landing pad and leucine landing pad. Both of these landing pads will replace the respective metabolic operons with our desired subcloned genetic elements. The leucine landing pad was constructed by a former member of the Ninfa lab, Dong Eun Chang and the arabinose landing pad was a part of our iGEM 2007 project, and was worked on by Alyssa Delke and Khalid Miri. In using these landing pads, we wish to limit the noise in our system in order to (hopefully) obtain more sustained oscillations than previous synthetic clocks have given.

LP