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 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 synthesis of various acid resistance proteins protect the drug of interest from the digestive assaults of the buccal cavity and acid-filled stomach. Once it reaches the intestine, gut microflora will strip away the E.ncapsulator's colanic acid coat allowing for release of the protein of interest.

Trehalose faciliates product storage. It does this by maintaining the protein in its correct three dimensional conformation, which would otherwise be disrupted by free radical attack and dessication.

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 with avirulence. We have additionally upregulated a third enzyme (rfal) to re-direct exopolysaccharide polysaccharide production to enhance the encapsulation of single cells (over and above colony encapsulation). This step further reduces virulence while enchancing pill functionality.

Finally, the two biosynthetic genes (OtsA & OtsB) code for the production of trehalose.

Module 2 Part i: Encapsulation

RcsB

Background:

RcsB is a transcription factor that forms part of the phosphorelay system. In response to membrane stress, RcsB is phosphorylated into its DNA binding form. In this state, it is able to both upregulate and downregulate a large number of genes.

RcsB upregulates the following genes:

ivy (Inhibitor of Vertebrate lysozyme)

Discovered in 2001 as the first bacterial lysozyme inhibitor. This Type-C lysozyme inhibitor resides in the periplasm.1

MilC (Membrane-bound lysozyme inhibitor of Type C lysozyme)

This is a lipoprotein that resides in the membrane. ¹

References

[http://www.ncbi.nlm.nih.gov/pubmed/19136591 The Rcs two-component system regulates expression of lysozyme inhibitors and is induced by exposure to lysozyme]

RcsB downregulates the following genes:

YgiV

Background:

In nature, the colanic acid synthesis phase occurs prior to biofilm formation. The latter process of biofilm formation is associated with the upregulation of a number of virulence factors. The transcription factor YgiV blocks the progression into biofilm formation by maintaining colanic acid production. Thus YgiV serves to increase acid resistance and decrease virulence.

Rfal

Background:

In the majority of E.coli, the enzyme Rfal joins the O-antigen to the membrane-bound lipid core molecule. Since the K-12 strain has an insertion mutation in the gene coding for O-antigen, the enzyme Rfal is free to join colanic acid to the lipid core.

Module 2 Part ii: Trehalose Production

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.