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Team:Imperial College London/M2
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In order to preserve the integrity of our protein of interest during storage of the E.ncapsulator, we decided to incorporate a device for trehalose production within our system. Trehalose is a disaccharide formed from two glucose molecules. Throughout nature, trehalose is associated with resistance to dessication and cold shock, and is naturally produced in Escherichia Coli. We hope that by upregulating the trehalose production pathways in E.coli we can increase trehalose concentrations within our cell, thereby conferring some resistance to protein degredation in our system. This would allow easy transport and storage of the final product.<br><br> | In order to preserve the integrity of our protein of interest during storage of the E.ncapsulator, we decided to incorporate a device for trehalose production within our system. Trehalose is a disaccharide formed from two glucose molecules. Throughout nature, trehalose is associated with resistance to dessication and cold shock, and is naturally produced in Escherichia Coli. We hope that by upregulating the trehalose production pathways in E.coli we can increase trehalose concentrations within our cell, thereby conferring some resistance to protein degredation in our system. This would allow easy transport and storage of the final product.<br><br> | ||
The trehalose coding region in E.coli consists of 2 genes, OtsA and OtsB - each coding for a different enzyme required for the conversion of glucose to trehalose. | The trehalose coding region in E.coli consists of 2 genes, OtsA and OtsB - each coding for a different enzyme required for the conversion of glucose to trehalose. | ||
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Revision as of 08:53, 3 September 2009
Contents |
Overview
What:
Module 2 is the encapsulation phase. The cell secretes an extracellular protective polysaccharide (colanic acid) which surrounds the cell. This forms a protective capsule that can withstand the acidic environment of the stomach. There is also the production of acid resistance proteins and storage metabolites (trehalose) which shield the protein of interest during passage through the stomach and facilitate product storage.
Why:
Colanic acid encapsulation and the synthesis of various acid resistance proteins protect the drug of interest from the digestive assaults of the buccal cavity and acid-filled stomach. Once the pill reaches the intestine, gut microflora will strip away the E.ncapsulator's colanic acid coat allowing for release of the protein of interest.
Trehalose preserves the integrity of our protein of interest during storage. It maintains the protein stability during dessication and cold shock, therefore conferring some resistance to protein degredation in our system. This would allow easy transport and storage of the final product.
When:
Module 2 is initiated following the completion of protein production (Module 1). It should be noted that Module 1 protein production continues at a lower 'maintenance level' throughout Module 2.
How:
Through the course of evolution, E.coli have equipped themselves with a multitude of defences to enable colanisation of the intestine. We are using two global transcription factors (RcsB & YgiV) to hijack this natural process in a way that maximises acid resitance. We have additionally upregulated a third enzyme (rfal) to enhance the encapsulation of single cells (over and above colony encapsulation). Finally, the two biosynthetic genes (OtsA & OtsB) code for the production of trehalose. These steps further reduce virulence while enchancing pill functionality.
