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. The cells also produce acid resistance proteins and storage metabolites (trehalose) which results in protein shielding and storage respectively.

Why:

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 strip away the E.ncapsulator's colanic acid coat allowing for release of 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 product allowing for easy transport and storage.

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. Our manipulation of endogenous pathways reduces virulence while enchancing pill functionality.