Team:Imperial College London/M2

From 2009.igem.org

(Difference between revisions)
Line 31: Line 31:
<b><i>The E.ncapsulatior</i></b> has been designed to be able to withstand  the rigours of stomach acid and freeze drying. This is achieved by the synthesis of the exopolysaccharide colanic acid and the cryoprotectant trehalose.  
<b><i>The E.ncapsulatior</i></b> has been designed to be able to withstand  the rigours of stomach acid and freeze drying. This is achieved by the synthesis of the exopolysaccharide colanic acid and the cryoprotectant trehalose.  
-
 
-
=Initiation:=
 
-
 
-
<b>Module 2</b> is triggered by a rise in levels of <i>cyclic Adenosine Mono Phosphate</i> (cAMP). This is because cAMP facilitates the dimerisation of the transcription factor CRP which induces transcription downstream of the PcstA promoter. The rise in cAMP is correlated with a fall in levels of the primary carbon source (glucose). Thus, by controlling the initial amount of glucose present in the system, we achieve a timer function for the start of <b>Module 2</b>.
 
-
 
-
<html>
 
-
<center>
 
-
<div class="highslide-gallery">
 
-
<a href="https://static.igem.org/mediawiki/2009/5/5b/CAMPModule2.png" class="highslide" onclick="return hs.expand(this, config1)">
 
-
<img src="https://static.igem.org/mediawiki/2009/5/5b/CAMPModule2.png" alt="" title="Click to enlarge" width="70%"/>
 
-
</a>
 
-
<div class="highslide-caption">
 
-
Module 2: Initiation
 
-
</div>
 
-
</div>
 
-
</center>
 
-
</html>
 
-
 
-
<html><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html>&nbsp; <b><i>About Module 2 temporal control.</i></b>
 
-
 
-
 
-
 
-
{{Imperial/09/Division}}
 
=Acid Resistance Theory:=
=Acid Resistance Theory:=

Revision as of 15:48, 18 October 2009

Contents

II09 Thumb m2.pngModule 2 - Encapsulsation

Module 2: Encapsulation

Module 2 Timeline

The E.ncapsulatior has been designed to be able to withstand the rigours of stomach acid and freeze drying. This is achieved by the synthesis of the exopolysaccharide colanic acid and the cryoprotectant trehalose.

Acid Resistance Theory:

By 'hacking' E.coli’s endogenous acid resistance pathways in three places we induced the formation of a safe colanic acid microcapsule that protects against the harsh acidic conditions of the stomach. Without encapsulation, our polypeptides would be denatured and degraded by stomach acid and digestive proteases respectively.

  About induced acid resistance.


The Encapsulation Triple Hack:

Module 2: The Triple Hack

Acid Resistance Results:

To characterise colanic acid encapsulation, we assembled the following testing constructs:

Module 2: Encapsulation Testing Construct

Module 2: Encapsulation Testing Construct


Freeze Drying Theory:

Freeze drying is the process by which a material is preserved via its dehydration at a very low temperature. Freeze drying our chassis would slow the decomposition of the polypeptide of interest and prevent the growth of any contaminants. Unfortunatly, freeze drying can seriously damage cell membranes and denature the internal polypeptides. To prevent this from occuring we have decided to upregulate the biosynthesis of the cryoprotectant trehalose. The genes OtsA and OtsB are required for trehalose production.

  About freeze drying & trehalose biosynthesis.


Module 2: Initiation


Freeze Drying Results:

To characterise the protective effect of trehalose against freeze drying, we assembled the following testing constructs:

Module 2: Trehalose Production Testing Construct

Unfortunatly, we did not have time to ligate together both OtsA and OtsB and were therefore unable to do any functional assays.


Finale:

The end of Module 2 occurs when the temperature of the culture is raised to 42 degrees centigrade. This temperature shift triggers the initiation of Module 3 in which the genome is deleted.

Project Tour



Module 2 Contents

Acid Protection
Freeze Drying
Genetic Circuit
Wet Lab
Modelling

Mr. Gene   Geneart   Clontech   Giant Microbes

Retrieved from "http://2009.igem.org/Team:Imperial_College_London/M2"