Team:Imperial College London/M3

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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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Module 2: Encapsulation
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[[Image: II09_gmo fears.jpg| right| 200px]]
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=<!--[[Image:II09_Thumb_m3.png|40px]]--><font size='5'><b>Module 3: Genome Deletion</b></font>=
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Module 3 is the final module of the system. It programs the E.ncapsulator to destroy its genetic material after encapsulation has finished. This prevents any possible pathogenic effects, and also allays health concerns of eating genetically modified organisms. <br>
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<br>
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10th International Symposium on the Biosafety of Genetically Modified Organisms (GM Biosafety Symposium)<br>
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[[Image:II09_transition_module3.jpg|center|400px]]
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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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[[Image:II09_Module3reusable.jpg|right|200px]]
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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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Module 3 consists of 2 parts: <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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1) Thermoinduction <br>
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<br>
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2) Killing by Restriction Enzymes<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>
<br>
<br>
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A distinct advantage of using restriction enzymes for our 'killing' mechanism is that the cell membrane is left intact afterwards, and the protein of interest will still be protected by the encapsulated cell.  This renders the bacterium no more than an inanimate shell containing our protein drug of choice. <br>
 
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[[Image: inanimate shell.jpg| center]]
 
<br>
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==Thermoinduction==
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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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[[Image: II09_temp increase.jpg| right]]
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[[Image:M3gci2.jpg|600px]]
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After Module 2 has been completed, genome deletion is triggered by raising the temperature. This is especially suitable since it is difficult for normal chemical induction to penetrate the colanic acid coating. <br>(see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]]) <br>
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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>
<br>
<br>
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The pLambda promoter and cI protein form a thermoregulatable system.  <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 producedThere 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>
<br>
<br>
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The cI protein represses the pLambda promoter at low temperatures. However, when temperature increases to 42 degrees celsius, the cI protein is denatured, and the lambda promoter is activated. <br>(see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]])<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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<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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==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>
 +
<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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<!--
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Module_3
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[[Image:II09_DNApic.png |right|250px]]
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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><br><br><b>Module 3</b> is the final module of our system. It is designed so that the cell is programmed to destroy it's genetic material after encapsulation has finished. <br><br>
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<br>
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After <b>Module 2</b> has been completed, <i>The E.ncapsulator</i> is programmed to 'genetically self-destruct.' The cell produces enzymes which specifically target and cut DNA sequences, in order to destroy all genetic material contained within the cell. One advantage of using these enzymes for our 'killing' mechanism is that the cell membrane is left intact afterwards, and the protein of interest will still be protected by the encapsulated cell.
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<br><br><br><br><br>
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More detail can be found [[Imperial_College_London/M3/Detail |here]].
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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><center><a href="https://2009.igem.org/Team:Imperial_College_London/M2"><img width=150px src="https://static.igem.org/mediawiki/2009/1/11/II09_M3ArrowLeft.png"></a>
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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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===Module 3 Contents===
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<html></center></html>
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[[Image:Mod3.jpg| 70px]]
 
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==Why==
 
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The <i>E.ncapsulator</i> requires the E. coli to be dead upon ingestion. This will prevent any transfer of genetic material between the bacterium and any gut microflora present, thereby avoiding any unexpected pathogenic effects. This is also especially important if the <i>E.ncapsulator</i> is to attain public acceptance, due to concerns over genetically modified organisms.
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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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==When==
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<html><table border="0" style="background-color:transparent;" width="100%">
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The thermally induced killing mechanism will only be triggered once encapsulation is complete.
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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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==How==
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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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Restriction enzymes found within bacteria and act as defense mechanisms against invading viruses. They work by recognising a certain DNA sequence of a few bases, and then cleaving the DNA strand. The <i>E.ncapsulator</i> is engineered to manufacture the restriction enzymes DpnII and TaqI when triggered, and these will cleave the genetic material within into fragments - thereby killing the cell.
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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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As a protective mechanism against DNA destruction due to basal levels of restriction enzyme production, we have made use of the native E. coli Dam methylase protection system. This methylates DNA, which means that only high levels of restriction enzyme (ie. upon trigger) will cleave the DNA.  
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<td width="20%"><center><a  
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is induced, rendering the bacterium no more than an inanimate shell containing our protein drug of choice.
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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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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

Mr. Gene   Geneart   Clontech   Giant Microbes