Team:Imperial College London/M3

From 2009.igem.org

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=Overview=
 
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==What:==
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/8/8d/II09_MapIndicator_Module3.png" class="highslide" onclick="return hs.expand(this, config1)" title="After thermoinduction, restriction enzymes are expressed that remove the genetic material">
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<img src="https://static.igem.org/mediawiki/2009/8/8d/II09_MapIndicator_Module3.png" alt="" title="Click to enlarge" width="75%"/>
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</a>
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<div class="highslide-caption">
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Module 2: Encapsulation
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</center>
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=<!--[[Image:II09_Thumb_m3.png|40px]]--><font size='5'><b>Module 3: Genome Deletion</b></font>=
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<br>
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[[Image:II09_transition_module3.jpg|center|400px]]
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The aim of <b>Module 3</b> is to neutralise any genetic material present within the cell, to ensure that the cells will not be able to proliferate and cause harm once ingested. The genetic material of the <i>E.ncapsulator</i> s cut up, leaving the cell membrane intact and the protein of interest contained within.
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<b>Module 3</b> is the final module of the system. <b><i>The E.ncapsulator</i></b> has successfully completed its job of protein production (module 1) and encapsulation (module 2).  Now, it needs to be prepared to be converted into a safe pill carrying the protein of interest. This is done by removing the genetic material which renders the cell inanimate.  <br>
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<br>
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[[Image:II09_Module3reusable.jpg|right|200px]]
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==Why==
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==Rationale==
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Module 3 acts as a <b>reusable</b> module for <b>removal of genetic material</b> without toxic effects.<br>
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<br>
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Removal of genetic material by the use of restriction enzymes prevents the accidental transfer of DNA to other gut microflora, which could lead to development of virulence. This module is a highly reusable for any chassis system where there is a need to remove genetic material after genes are expressed. <br>
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<br>
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Our pill is to be <b>consumed</b> within the human body.  This rules out the <b>toxin-generating</b> methods to induce cell death. Restriction enzymes are the preferred method for inducing cell death as they ar relatively harmless outside of the cell. <br>
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<br>
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<br>
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The E.ncapsulator requires that the E. coli should be dead upon ingestion. This will prevent any transfer of genetic material between bacterium and host, and will also help to allay fears that people may have with regards to ingesting live bacteria.
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==Theory==
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===Engineering cell death===
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Due to the possible <b>pathogenicity and health concerns</b>, cell death must occur before the pill is ready for consumption.  Therefore, the method chosen needs to be foolproof and have <b>failsafe mechanism</b>. <br>
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<br>
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==When==
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[[Image:M3gci2.jpg|600px]]
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<br><br>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Genetic
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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><b>&nbsp; About our genetic circuit</b>
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<br><br>
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<br>
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Under the control of a thermoinducible promoter system ([http://partsregistry.org/Part:BBa_K098995 K098995]), when the temperature is raised, the promoter is activated and restriction enzymes are produced.  There is a safeguard here as the temperature of the human body is around 37°C, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body. <br>
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<br>
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The restriction enzymes DpnII ([http://partsregistry.org/Part:BBa_K200009 K200009]) and TaqI ([http://partsregistry.org/Part:BBa_K200010 K200010]) are produced.  This duplicity of restriction enzymes ensures that even when one enzyme becomes mutated and dysfunctional, the other restriction enzyme still works well by itself. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested.
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<br>
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<br>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/RestrictionEnzymes
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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><b>&nbsp; About Restriction Enzymes</b>
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<br><br>
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<br>
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Dam methylase ([http://partsregistry.org/Part:BBa_K200001 K200001]) is constitutively produced at a low amount.  This prevents leaky expression of restriction enzymes from damaging the genome prematurely. Consequently, a balance exists between Dam methylation and restriction enzyme activity.  <br><br>
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The killing mechanism is only to be triggered once encapsulation is complete. This caveat brings with it certain restrictions in terms of trigger mechanisms that could be adopted in our system; the presence of the colanic acid capsule means chemical or light triggers would not be as efficient as when the capsule is lacking. To overcome this, genomic neutralisation is induced by thermoinduction.
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation
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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><b>&nbsp; About Methylation</b>
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==How==
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==Results==
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===Wet Lab===
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[[Image:II09 DpnII Digest.png|right|400px]]
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The activity of the restriction enzymes is critical to module 3.  We have tested this using a genomic digest assay. <br>
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<br>
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The restriction enzymes DpnII and TaqI are shown to cut genomic DNA into small fragments, shown on the right by a smear of bands.  We have further tested the restriction enzymes in DNA which have been methylated by Dam enzymes and shown that there is essentially no cleavage at low concentrations of restriction enzymes. <br>
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<br>
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The bacterial membrane is left intact so as not to disrupt the structure of the colanic acid layer. Any killing mechanism that completely destroys the bacterium would defeat the purpose of having a self-encapsulating drug production and delivery system. With this in mind, a strategy was devised that takes advantage of restriction enzymes DpnII and TaqI and the native E. coli dam methylase protection against these restriction enzymes. The dam methylation provides protection to the bacterium during drug production and encapsulation stages.  
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Module_3
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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 our wet lab results</b>
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<br>
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The λcI gene inhibits the λcI promoter under which DpnII and TaqI are controlled. Upon increasing temperature to 37°C - 40°C the λcI gene undergoes a conformational change and is no longer able to bind the λcI promoter. The promoter is then free to transcribe DpnII and TaqI and cleavage of the E. coli genome is induced, rendering the bacterium no more than an inanimate shell containing our protein drug of choice.
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[[Image:M3s.6.png|right]]
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<br>
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===Dry Lab===
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We have also attempted to link our restriction enzymes with cell death using a model.<br>
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<br>
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The population increase is initially exponential as the restriction enzymes have a delay in production.  As the restriction enzymes accumulate in the cell, the cell growth starts to slow down.  If the lambda cI promoter is strong enough, killing rate will greatly exceed cell division rate, and there will be an exponential decrease in cell population. <br>
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<br>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Genome_deletion
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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 our dry lab results</b><br>
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<br>
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===Results summary===
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We have shown that cells can be protected from low concentrations of the restriction enzymes DpnII and TaqI by Dam methylation, and how the cell population rapidly decreases with thermoinduction of restriction enzymes. <br>
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<br>
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<html><center></html>
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===Project Tour===
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/Thermoinduction"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/TIL.jpg"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/TemporalControlR.jpg"></a></center>
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</html>
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<br>
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<hr>
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===Module 3 Contents===
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<html></center></html>
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3/RestrictionEnzymes"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage5.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Homepageimage3.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Genetic"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_geneticcircuit1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Wetlab/Results#Module_3"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"></a><html><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Genome_deletion"><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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<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/M3/RestrictionEnzymes"><b>Restriction Enzymes</b></a></center></td>
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<td width="20%"><center><a href="/Team:Imperial_College_London/M3/DamMethylation"><b>DAM Methylation</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Genetic"><b>Genetic Circuit</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/M3/Wetlab/Results#Module_3"><b>Wet Lab</b></a></center></td>
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<td width="20%"><center><a
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href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Genome_deletion"><b>Modelling</b></a></center></td>
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<td width="1%"></td>
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</tr></table></html>
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{{Imperial/09/TemplateBottom}}

Latest revision as of 03:54, 22 October 2009


Contents

Module 3: Genome Deletion


II09 transition module3.jpg

Module 3 is the final module of the system. The E.ncapsulator has successfully completed its job of protein production (module 1) and encapsulation (module 2). Now, it needs to be prepared to be converted into a safe pill carrying the protein of interest. This is done by removing the genetic material which renders the cell inanimate.

II09 Module3reusable.jpg

Rationale

Module 3 acts as a reusable module for removal of genetic material without toxic effects.

Removal of genetic material by the use of restriction enzymes prevents the accidental transfer of DNA to other gut microflora, which could lead to development of virulence. This module is a highly reusable for any chassis system where there is a need to remove genetic material after genes are expressed.

Our pill is to be consumed within the human body. This rules out the toxin-generating methods to induce cell death. Restriction enzymes are the preferred method for inducing cell death as they ar relatively harmless outside of the cell.


Theory

Engineering cell death

Due to the possible pathogenicity and health concerns, cell death must occur before the pill is ready for consumption. Therefore, the method chosen needs to be foolproof and have failsafe mechanism.

M3gci2.jpg

  About our genetic circuit


Under the control of a thermoinducible promoter system ([http://partsregistry.org/Part:BBa_K098995 K098995]), when the temperature is raised, the promoter is activated and restriction enzymes are produced. There is a safeguard here as the temperature of the human body is around 37°C, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body.

The restriction enzymes DpnII ([http://partsregistry.org/Part:BBa_K200009 K200009]) and TaqI ([http://partsregistry.org/Part:BBa_K200010 K200010]) are produced. This duplicity of restriction enzymes ensures that even when one enzyme becomes mutated and dysfunctional, the other restriction enzyme still works well by itself. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested.

  About Restriction Enzymes


Dam methylase ([http://partsregistry.org/Part:BBa_K200001 K200001]) is constitutively produced at a low amount. This prevents leaky expression of restriction enzymes from damaging the genome prematurely. Consequently, a balance exists between Dam methylation and restriction enzyme activity.

  About Methylation

Results

Wet Lab

II09 DpnII Digest.png

The activity of the restriction enzymes is critical to module 3. We have tested this using a genomic digest assay.

The restriction enzymes DpnII and TaqI are shown to cut genomic DNA into small fragments, shown on the right by a smear of bands. We have further tested the restriction enzymes in DNA which have been methylated by Dam enzymes and shown that there is essentially no cleavage at low concentrations of restriction enzymes.

  About our wet lab results

M3s.6.png


Dry Lab

We have also attempted to link our restriction enzymes with cell death using a model.

The population increase is initially exponential as the restriction enzymes have a delay in production. As the restriction enzymes accumulate in the cell, the cell growth starts to slow down. If the lambda cI promoter is strong enough, killing rate will greatly exceed cell division rate, and there will be an exponential decrease in cell population.

  About our dry lab results

Results summary

We have shown that cells can be protected from low concentrations of the restriction enzymes DpnII and TaqI by Dam methylation, and how the cell population rapidly decreases with thermoinduction of restriction enzymes.

Project Tour



Module 3 Contents


Restriction Enzymes
DAM Methylation
Genetic Circuit
Wet Lab
Modelling

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