Team:Imperial College London/M3/RestrictionEnzymes

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=[[Image:II09_Thumb_m3.png|40px]]<font size='5'><b>Module 3: Genome Deletion Overview</b></font>=
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==Restriction Enzymes==
==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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Restriction enzymes will cleave DNA at particular recognition sites. This leads to multiple double-stranded breakages in DNA, which are unlikely to be repaired in time and will subsequently result in cell death.  Restriction enzymes are therefore highly toxic to the cell, where even minute amounts can induce destruction of the genome.  <br>
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see [[Team:Imperial_College_London/Temporal_Control/Thermoinduction| thermoinduction under temporal control]])<br>
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<br>
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In our system, we have made use of these properties of restriction enzymes.  The restriction enzymes DpnII and TaqI are produced. These serve to effectively remove the genetic material from our <i>E.ncapsulator</i>. <br>
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<br>
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DpnII and TaqI are 4 base cutters, specifically targetting and cutting the sequences GATC and TCGA respectively. <br>
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<br>
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[[Image: II09_taq&dpn2 digestion.jpg]]
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<br>
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<br>
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4 base cutters were chosen as they have a higher frequency of cleavage.  Assuming equal distribution of nucleotides, each nucleotide has an equal probabiliy of occurring at any site.  <br>
 +
<br>
 +
The probability of cleavage for a 4-base long sequence is : <br>
 +
<br>
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1/4 x 1/4 x 1/4 x 1/4 = (1/4)<sup>4</sup> <br>
 +
<br>
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This means that on average the four cutter will cut every 256 base pairs.  Given that the genome of E.coli is around 4 million base pairs, it will become totally digested. <br> This ensures that the genetic material contained within the cell will all be destroyed, including any inserted DNA. <br>
 +
<br>
 +
[[Image: II09_RE activity.jpg| right]]
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Having 2 different restriction enzymes is part of a failsafe engineering design.  These restriction enzymes have similar recognition sequences, allowing for an overlap in cleavage capabilities.  When both restriction enzymes are expressed, there is comprehensive cutting, but 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. <br>
<br>
<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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<!--
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[[Image: inanimate shell.jpg| center]]
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M3/REcutting"><img style="vertical-align:bottom;" width=90px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html><br>&nbsp; Click here for more information regarding frequency of cutting by 4 cutters
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<!--[[Image: inanimate shell.jpg| left | size=400px]]-->
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A distinct advantage of using restriction enzymes for our 'killing' mechanism is that the the genetic material is removed, but the cell membrane is left intact.  Therefore, the protein of interest will still be protected by the encapsulated cell.  This renders the bacterium no more than an inanimate shell loaded with our protein drug of interest. <br>
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==References==
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[http://www.rebase.neb.com| Rebase]
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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.
<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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===Module 3 - Genome Deletion===
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<center><b>Module 3 - Genome Deletion </b></center>  
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3"><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/DamMethylation"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Homepageimage3.png"></a><a  
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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  
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Genetic"><img style="vertical-align:bottom;" width="23%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Homepageimage3.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/M3/Wetlab"><img style="vertical-align:bottom;" width="23%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Homepageimage4.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Modelling"><img style="vertical-align:bottom;" width="23%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Homepageimage5.png"></a><html><a  
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href="https://2009.igem.org/Team:Imperial_College_London/M3/Wetlab"><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/M3/Modelling"><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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href="https://2009.igem.org/Team:Imperial_College_London/M3/Results"><img style="vertical-align:bottom;" width="27%" src="https://static.igem.org/mediawiki/2009/2/20/II09_WorldMap.png"></a><center></html>
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<html><table border="0" style="background-color:transparent;" width="100%">
<html><table border="0" style="background-color:transparent;" width="100%">
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<td width="25%"><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="25%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/M3/Wetlab"><b>WetLab</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3"><b>Module 3 Overview</b></a></center></td>
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<td width="25%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Modelling"><b>Modelling</b></a></center></td>
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<td width="25%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3/Results"><b>Results</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"><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/M3/Modelling"><b>Modelling</b></a></center></td>
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<td width="1%"></td>
<td width="1%"></td>
</tr></table></html>
</tr></table></html>
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/M3#Module_3_Contents"><img width=150px src="https://static.igem.org/mediawiki/2009/1/10/II09_TourArrow.png"></a>
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</html>
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Latest revision as of 21:24, 17 October 2009

Contents

II09 Thumb m3.pngModule 3: Genome Deletion Overview

Restriction Enzymes

Restriction enzymes will cleave DNA at particular recognition sites. This leads to multiple double-stranded breakages in DNA, which are unlikely to be repaired in time and will subsequently result in cell death. Restriction enzymes are therefore highly toxic to the cell, where even minute amounts can induce destruction of the genome.

In our system, we have made use of these properties of restriction enzymes. The restriction enzymes DpnII and TaqI are produced. These serve to effectively remove the genetic material from our E.ncapsulator.

DpnII and TaqI are 4 base cutters, specifically targetting and cutting the sequences GATC and TCGA respectively.

II09 taq&dpn2 digestion.jpg

4 base cutters were chosen as they have a higher frequency of cleavage. Assuming equal distribution of nucleotides, each nucleotide has an equal probabiliy of occurring at any site.

The probability of cleavage for a 4-base long sequence is :

1/4 x 1/4 x 1/4 x 1/4 = (1/4)4

This means that on average the four cutter will cut every 256 base pairs. Given that the genome of E.coli is around 4 million base pairs, it will become totally digested.
This ensures that the genetic material contained within the cell will all be destroyed, including any inserted DNA.

II09 RE activity.jpg

Having 2 different restriction enzymes is part of a failsafe engineering design. These restriction enzymes have similar recognition sequences, allowing for an overlap in cleavage capabilities. When both restriction enzymes are expressed, there is comprehensive cutting, but 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.




A distinct advantage of using restriction enzymes for our 'killing' mechanism is that the the genetic material is removed, but the cell membrane is left intact. Therefore, the protein of interest will still be protected by the encapsulated cell. This renders the bacterium no more than an inanimate shell loaded with our protein drug of interest.

References

Rebase



Mr. Gene   Geneart   Clontech   Giant Microbes