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.

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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.

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
 * The Rcs two-component system regulates expression of lysozyme inhibitors and is induced by exposure to lysozyme

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.