Team:Imperial College London/M2

Overview

WHAT

Module 2 is a distinct stage that involves the production of an extracellular protective polysaccharide (colanic acid), acid resistance proteins, and storage metabolites.

WHEN

Module 2 is initiated following the completion of protein production (Module 1) and prior to genomic neutralisation (Module 3). It should be noted that Module 1 protein production will continue throughout Module 2 but at a heavily reduced rate such that additional resources can be devoted to Module 2 activity.

WHY

Abiotic resistance faciliates product storage. To achieve this, trehalose is produced during Module 2 to help maintain structural integrety during the freeze drying process. Trehalose additionally, protects the internal 'protein payload' from free radical attack.

Biotic resistance faciliates delivery passed the digestive assalts of the buccal cavity and acid-loaded stomach. The exopolysaccharide colanic acid and resistance proteins protects the E.ncapsulator against these harsh conditions. Once in the intestine, gut microflora strip the E.ncapsulator of its colanic acid coating paving the way for 'payload release'.

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 transcription factors (RcsB & ) to hijack this natural process in a way that combines confers acid resitance with avirulence. We have additionally upregulated a third enzyme (Waal ligase) 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, two genes (OtsA & OtsB) code for the production of trehalose.

Module 2 of our system consists of 2 parts:
i) An encapsulation mechanism - through which our cells secrete an exopolysaccharide capsule, to protect the cell from the harsh conditions encountered in the stomach.
ii) A storage mechanism - through which a disaccharide (trehalose) is produced within the cell, with the intention of conferring our protein of interest with some resistance to cold shock and dessication.

Module 2 Part i: Encapsulation

In order to deliver our protein of interest to the gut, we must find some way of conferring our cells with some ability to withstand the harsh conditions present within the stomach.

Escheria coli naturally has a mechanism for withstanding these conditions, allowing them to survive through to the gut. This mechanism consists of secretion of a 'slime capsule,' which allows the cells to resist the highly acidic environment of the stomach. This 'slime capsule' mainly consists of secreted exopolysaccharides, such as colanic acid. By upregulating these genes that are naturally present, this will give our cells the ability to 'auto-encapsulate' - to secrete this slime capsule when required, and allowing our cells to bypass the stomach.

There are several genes required for synthesis of colanic acid. RcsB, WaaL, B3023

This image shows a cross section of our cells, consisting of our protein of interest within, surrounded by ...

RcsB

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:

Module 2 Part ii: Trehalose Production