Team:Imperial College London/M3

From 2009.igem.org

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Under the control of a thermoinducible promoter (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 degrees, so that even if the bacteria are not killed by the heat pulse, they will be killed after they enter the human body. <br>
Under the control of a thermoinducible promoter (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 degrees, 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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The restriction enzymes DpnII (K200009) and TaqI (K200010) are produced.  This duplicity of restriction enzymes ensures that even if one enzyme is inactivated, there will still be another restriction enzyme cut frequently in the genome, digesting the entire genetic material. Dam methylase (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 restriction enzymes DpnII (K200009) and TaqI (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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<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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Dam methylase (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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<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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<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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Revision as of 21:53, 19 October 2009

Please delete as completed.
Module 3 feedback from todays session:

1) Bold important sentences/words
2) Presentation is different to the previous pages - restructure
3) 3 learn more tabs in a row - yucky
4) If no data exists - say that as the wiki is being frozen we haven't added the data but will have it in time for the Jamboree.
5) Have a rationale section.
6) Add what teams can reuse from this module.
7) Have a conclusion of the page at the end - couple of lines.
8) Link to the relevant Wet Lab section
9) Remove !'s in learn mores


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 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.

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.

M3gci.jpg

Under the control of a thermoinducible promoter (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 degrees, 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 (K200009) and TaqI (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 (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

  About our genetic circuit!






Our results

Wetlab

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.

 <i> About our wetlab results!

M3s.6.png


Drylab

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 drylab results!



Project Tour



Module 3 Contents


Restriction Enzymes
DAM Methylation
Genetic Circuit
Wet Lab
Modelling


Mr. Gene   Geneart   Clontech   Giant Microbes