Team:Imperial College London/M3

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=<!--[[Image:II09_Thumb_m3.png|40px]]--><font size='5'><b>Module 3: Genome Deletion</b></font>=
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<img src="http://i691.photobucket.com/albums/vv271/dk806/MainOverviewPicM3.jpg" usemap="#MAP91791" border="0" width="75%" title="Overview picture of the E.ncapsulator">
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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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[[Image:II09_Module3flow2.jpg|right|250px]]
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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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<b>Module 3</b> is the final module of the system. <b><i>The E.ncapsulator</i></b> has successfully completed its job of polypeptide production and encapsulation.  Now, it needs to be prepared to be converted into a safe pill carrying the polypeptide 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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====Reusable module for removal of genetic material====
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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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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 module for any chassis system where there is a need to remove genetic material after genes are expressed. <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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====The genetic circuit====
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==Theory==
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[[Image:M3gci.jpg|600px]]
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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 consumptionTherefore, the method chosen needs to be foolproof and have <b>failsafe mechanism</b>.  <br>
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Under the control of a thermoinducible promoter (K098995), when the temperature is raised to 42°C, the promoter is activated, and the restriction enzymes DpnII (K200009) and TaqI (K200010) are produced.    These restriction enzymes cut frequently in the genome, digesting the entire genetic material.  Dam methylase (K200001) is constitutively produced at a low amountThis 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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[[Image:M3gci2.jpg|600px]]
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<br><br>
<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Genetic
<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><i><b>&nbsp; About our genetic circuit!</b>
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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>
 +
<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>
<br>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/RestrictionEnzymes
<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/RestrictionEnzymes
"><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>  
"><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>
<br>
<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>
<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation
<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/DamMethylation
"><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>
"><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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====Our results====
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<u>Wetlab</u>
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[[Image:II09 DpnII Digest.png|right|400px]]
[[Image:II09 DpnII Digest.png|right|400px]]
The activity of the restriction enzymes is critical to module 3.  We have tested this using a genomic digest assay. <br>
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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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Module_3
<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/II09_Learnmore.png"></a></html>&nbsp; about our wetlab results!
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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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<u>Drylab</u>
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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>
We have also attempted to link our restriction enzymes with cell death using a model.<br>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Genome_deletion
<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/II09_Learnmore.png"></a></html>&nbsp; about our drylab results!
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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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<b>The system</b>
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Under control of a thermoinducible promoter, when the temperature is raised to 42°C, the restriction enzymes DpnII and TaqI are produced.  These restriction enzymes cut frequently in the genome, digesting the entirity of the genetic material.  Without its genetic material, cell death is also naturally induced.  However, restriction enzymes are highly toxic to the cell, where even minute amounts can induce destruction of the genome.  Therefore, the native Dam methylation system is used to prevent leaky levels of restriction enzymes from killing the cell prematurely. The restriction enzyme cleavage properties and Dam methylation protection form an interesting system for further study. <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>
<br>
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<!--This prevents any possible pathogenic effects, and also allays health concerns of eating genetically modified organisms.  -->
 
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Stomach"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html><br><br>&nbsp; About the ethical implications of live organisms.
 
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==Restriction Enzymes==
 
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<!--After Module 2 has been completed, genome deletion is triggered by raising the temperature. <br>
 
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see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]])<br>
 
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The cell then produces the [[Team:Imperial_College_London/M1/RE| restriction enzymes]] DpnII and TaqI. 
 
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Restriction enzymes DpnII and TaqI specifically target and cut short 4 base DNA sequences.  With a high frequency of cutting, the genetic material contained within the cell will all be destroyed, including any inserted DNA. <br>
 
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[[Image: II09_cut dna.jpg| center]]
 
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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]]
 
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<br>
 
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Restriction_Enzymes"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html><br><br>&nbsp; about the restriction enzymes TaqI and DpnII.
 
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<!--{{Imperial/09/Division}}
 
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==Dam methylation==
 
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To protect against DNA destruction due to basal levels of restriction enzyme production, we have made use of the native E. coli [[Team:Imperial_College_London/M1/Dam| Dam methylase protection system]]. This methylates DNA.  Therefore, only high levels of restriction enzyme (ie. after thermal triggering) will cleave the DNA.<br>
 
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Dam"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html><br><br>&nbsp; about the Dam methylation Defense Mechanism -->
 
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==Thermoinduction==
 
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[[Image: II09_temp increase.jpg| right]]
 
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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>
 
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<br>
 
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The pLambda promoter and cI protein form a thermoregulatable system.  <br>
 
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see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]])<br>
 
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[[Image:II09_DNApic.png |right|250px]]
 
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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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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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<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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[[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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==When==
 
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The thermally induced killing mechanism will only be triggered once encapsulation is complete.
 
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==How==
 
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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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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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is induced, rendering the bacterium no more than an inanimate shell containing our protein drug of choice.
 
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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

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