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Team:Imperial College London/M2
From 2009.igem.org
Module 2 Overview
Aside from aiding in pill manufacture, encapsulation (Module 2) allows the safe passage of polypetides through the stomach into the intestine. The encapsulation process involves the production of a physical barrier, acid resistance proteins and the preservative trehalose. Without encapsulation, our polypeptides would be denatured and degraded by stomach acid and stomach proteases respectively.
To learn more about the harsh conditions of the stomach, please click on The E.ncapsulator 
Encapsulation Rationale
In nature, encapsulation pathways such as spore formation, aliginate biosynthesis and colanic acid production all share one common feature: they require a large number of genes. For this reason, we decided that the best way encapsulate our chassis was via the modulation of an existing pathway.
E.coli naturally produces a harmless acid resistant polymer known as colanic acid. By tapping into the pathway that initiates colanic acid biosynthesis, we can turn on its production via the modulation of a gene called RcsB.
In nature, colanic acid acts as a binding agent between individual cells over which a biofilm can be formed. While colanic acid itself is harmless, biofilm formation is associated with the production of a number of virulence factors. To prevent biofilm formation from occuring, we have tapped into a second pathway such that our cells become locked into colanic acid production. The gene responsible for preventing biofilm formation is a transcription factor called YgiV.
In nature, colanic acid is associated with but not attached to the cell surface. To facilitate whole cell encapsulation, we have modified a third pathway to fix the colanic acid to the surface of the cell. This involves an enzyme called Rfal.
To learn more about RcsB, YgiV and Rfal, please click on The E.ncapsulator
Freeze Drying
Cells can be stored for extended periods of time by dehydration. However, under such conditions the integrety of both a cell's membrane and intracellular polypeptides can be compromised.
Trehalose is a disaccharide formed from two glucose molecules that provides resistance to dessication. While trehalose is naturally produced in E.coli, we hope that by upregulating its production, we can confer additional resistance to freeze drying. This would allow easy transport and storage of the final product.
OtsA and OtsB are the two genes required for trehalose production. We hope that by introducing additional copies of these genes on a plasmid we can boost trehalose production.
