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Team:Imperial College London/M2
From 2009.igem.org
Please delete as completed.
Module 2 feedback from todays session:
1) Remind reader of constraints of the system - why we need protection.
2) More detail and add references. Keep it technical so understandable to all.
3) Justify use of CA & compare to the alternatives - say it meets our requirements.
4) Content is good - just need to restructure it.
5) Encapsulation triple hack - Why? Need to introduce this.
6) Results: Need pics of plates - CHARLES TO BRING IN CAMERA! Growth assay - tomorrow?!
7) Trehalose - need this but don't have data for freeze drying...
8) Remove finale - keep the arrow structure like the other pages
9) Why 42 degrees and when? Explain. Say once got enough protection... blah
10) Link all constructs to registry.
11) 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.
12) Have a rationale section.
13) Add what teams can reuse from this module.
14) Have a conclusion of the page at the end - couple of lines.
Contents |
Module 2 - Encapsulsation
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.
The need for acid resistance:
To ferry polypeptides through the stomach, each of our microcapsules must withstand a heated protease–rich acid bath. Let there be no illusions, this is no mean feat. The stomach is a highly evolved microbe–mincer that few chassis have the potential to withstand. What is more, since our microcapsules are inanimate, we cannot rely on any of the active acid–resistance strategies that living bacteria are able to deploy.
About proteolysis.
This point is illustrated one of our experiment shown below:
Constitutive GFP producing E.coli cells were exposed to differing concentrations of acid for 30 minutes. The reduction in fluoresence is indicative of cell lysis and the subsequent acid-induced denaturing of GFP.
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:
Acid Resistance Results:
To characterise colanic acid encapsulation, we assembled the following testing constructs:
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.
Freeze Drying Results:
To characterise the protective effect of trehalose against freeze drying, we assembled the following testing constructs:
Unfortunatly, we did not have time to ligate together both OtsA and OtsB and were therefore unable to do any functional assays.
Project Tour
Module 2 Contents
