Imperial College London/M2/Detail

The primary function of The E.ncapsulator is to allow delivery of polypeptide pharmaceuticals to the gut. This requires passing through the highly acidic conditions present within the stomach, before being released into the small intestine.

E.coli has several native mechanisms involved for production of a capsule. Colanic acid encapsulation and the synthesis of various acid resistance proteins protect the protein of interest from the digestive assaults of the buccal cavity and acid-filled stomach. Once the pill reaches the intestine, gut microflora will digest the E.ncapsulator's colanic acid coat, releasing the protein of interest.

Trehalose preserves our protein of interest in its correct conformation in response to dessication. Thus trehalose allows for the freeze drying of the pill, and this will allow easy transport and storage.

Encapsulation is initiated following the completion of protein production (Module 1). It should be noted that the protein production of our compound of interest continues at a lower 'maintenance level' throughout Module 2.

E.coli is naturally equipped with a multitude of defences to enable colonisation of the intestine. The E.ncapsulator is programmed to upregulate two global transcription factors (RcsB & YgiV) to hijack this natural process to maximise 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. Our manipulation of endogenous pathways reduces virulence while enchancing pill functionality.

An important consideration when designing the specifications of the E.ncapsulator was the ability to store the cells for extended periods of time. This could be achieved by dehydrating the cells. However, normally under such conditions there poses a problem to maintaining the integrity of the proteins within the cells. This is problematic for us, as this could lead to breakdown of our protein of interest.

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.

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.