Team:Imperial College London/M3

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<li>[[IGEM:IMPERIAL/2009/M3/Modelling/overview|Overview]]</li>
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<li>[[IGEM:IMPERIAL/2009/M3/Modelling/analysis|Models]]</li>
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<li>[[IGEM:IMPERIAL/2009/M3/Modelling/simulations|Simulations]]</li>
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<li>[[IGEM:IMPERIAL/2009/M3/Modelling/old|Old]]</li>
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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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Module 2: Encapsulation
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=<!--[[Image:II09_Thumb_m3.png|40px]]--><font size='5'><b>Module 3: Genome Deletion</b></font>=
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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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<br>
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[[Image:II09_Module3reusable.jpg|right|200px]]
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=Background=
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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>
<br>
<br>
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The E. coli is killed after it has successfully produced PAH or cellulase, has encapsulated itself in colanic acid and produced trehalose to increase stability of PAH or cellulase during storage. Killing is necessary to make the pill non-toxic or pathogenic, and hence safe for human consumption. This self killing mechanism will be activated by an increase in temperatures from 28c to 42c. Therefore, this module will enable us to kill the E. coli after protein production and encapsulation has finished.
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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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==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:M3gci2.jpg|600px]]
<br><br>
<br><br>
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[[Image:m3gc.jpg]]
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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>
<br><br>
<br><br>
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Initially, the lambda cI promoter is repressed by the protein cI that is produced constitutively under the strong promoter J23114. At 28°C, functional protein cI will bind to the lambda cI promoter to repress it. Restriction enzymes DpnII and TaqI will not be produced.  
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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>
 +
<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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When there is an increase in temperature (from 28°C to 42°C <cite>1</cite><cite>2</cite>), a change in conformation of the cI protein occurs. Now, protein cI will no longer be able to bind to the lambda cI promoter to repress it. In other words, there will be a de-repression of lambda cI promoter, and it will be activated, to produce restriction enzymes DpnII and TaqI. Restriction enzymes will digest the genetic material of the E. coli cells and cut the DNA, leading to cell death.
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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>
 +
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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<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 Dam methylase, which is produced constitutively by a weak promoter J23103, will prevent cell death for redundant basal levels of production of restriction enzymes. This will ensure that unwanted cell death will not occur before we trigger this module. In this way, we can allow E. coli to produce as much PAH or cellulase as possible, with ample time for encapsulation, before we trigger cell death manually.  
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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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Dam methylase works by transferring a methyl group to the N6 position of the adenine residues in the sequence GATC[[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_cpg_methylation.asp 3]]. As a result, restriction enzymes cannot recognise their intended cleavage site. This means that DNA will not be cleaved in the presence of Dam methylase and E. coli cells will not die. As it competes with restriction enzymes for DNA, at low restriction enzymes concentrations, Dam methylase will be able to methylate DNA and protect it. However, as the concentration of restriction enzymes increase at higher temperatures, the amount of constitutively produced Dam methylase will no longer be sufficient to protect against cell death, and cell killing occurs.  
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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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=Goals=
 
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We aim to:
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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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*Explore how temperature correlates to restriction enzyme concentration, and see how it affects the population of live cells, so as to characterise the effects of temperature on cell death.
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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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*Develop a model of the number of dead cells as this correlates to our live and dead cells assay. (hyperlink to assay). From this, we can monitor the rate of killing and perform data analysis to obtain the necessary parameters.
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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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*Model for the effects of Dam methylase in protection against cell death. We want to know how much Dam methylase will be necessary to prevent cell death for basal production of restriction enzymes.
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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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In summary, our model will allow us to characterise the effects of temperature on the parallel production of restriction enzymes DpnII and TaqI, that are required to kill E. coli. We can also characterise the extent Dam methylase can protect E. coli against cell death. (not yet done)
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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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<br>
 
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<biblio>
 
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#1 pmid=15652426
 
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#2 pmid=10235623
 
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</biblio>
 
{{Imperial/09/TemplateBottom}}
{{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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