Module 2 - Encapsulsation Overview

Encapsulation Rationale

To ferry polypeptides through the stomach, each of our microcapsules must withstand the rigours of a heated protease–rich acid bath. Let there be no illusions, this is no mean feat. The stomach is a highly evolved microbe–mincer that few chassis have the potential to withstand. What is more, since our microcapsules are inanimate, we cannot rely on any of the active acid–resistance strategies that living bacteria are able to deploy.

To tackle this seemingly insurmountable problem we adopted a two phase approach.

Phase 1: Identify a suitable chassis with the genotypic potential for acid–resistance.

Phase 2:Manipulate endogenous acid resistance pathways to control the acid resistant phenotype.

Phase 1:

Our rationale for looking for natural sources of acid resistance is that it is easier to hack existing pathways than to transfer large numbers of genes into a different chassis with a dissimilar genetic background.

Based on natural sources of acid resistance, Lactobacillus, E.coli and B.subtilis were shortlisted as potential chassis.

Registry Code

Type

Sequence Description

Coding

RcsB is a receiver protein which acts as a positive regulator of a number of genes including capsule genes responsible for colanic acid production.

Coding

Dam (DNA Adenine Methylase) The methylase encoded by the dam gene ([http://en.wikipedia.org/wiki/Dam_(methylase) Dam methylase]) transfers a methyl group from S-adenosylmethionine to the N6 position of the adenine residues in the sequence GATC, this protects the DNA from cleavage.

Coding

Colanic acid global regulator ygiV (B3023) increases the production of colanic acid further in conjunction with RcsB by acting as a repressor for mcbR/yncC promoter. YncC/mcbR normally repress colanic acid overproduction so as to increase biofilm formation.

Coding

Waal Ligase is an enzyme responsible for the ligation of an [http://en.wikipedia.org/wiki/O_antigen#O-antigen O-antigen] to the core [http://en.wikipedia.org/wiki/Oligosaccharide oligosaccharide] in the Gram-negative bacterium's outer membrane.

Coding

B.subtilis: Pros: 1. Can form a highly acid–resistant spore. Cons: 1. Sporulation follows starvation which could result in the destruction of the polypeptides which we are seeking to deliver. 2. Safety. 3. Difficult to work with

E.coli Pros: 1. Acid a broad range of acid resistance strategies 2. Probiotic K12 strains available Cons: 1. Can form biofilms which are associated with virulence

Lactobacillus: Pros: 1. Acid resistant 2. Safe

Cons: 1. Difficult to work with

Based on these criteria, we decided that E.coli was the most suitable chassis.

Phase 2:

We hacked E.coli’s acid resistance module in three places to achieve the production of a safe acid resistant capsule.

Acid Resistant Polymer – Colanic acid: E.coli naturally produces a harmless acid–resistant polymer known as colanic acid. Colanic acid is a polymer of glucose, galactose and glucuronic acid. By tapping into the pathway that initiates colanic acid biosynthesis, we can turn on its production via the modulation of a transcription factor encoded by gene called RcsB.

About RcsB

Safety – Biofilm prevention: In nature, colanic acid acts as a binding agent between individual cells over which a biofilm can be formed. While colanic acid itself is harmless, biofilm formation is associated with a number of virulence factors. To prevent biofilm formation from occurring, we have tapped into a second pathway such that our cells become locked into colanic acid production. The gene responsible for preventing biofilm formation is a transcription factor encoded by a gene called YgiV.

About YgiV

Microencapsulation – Colanic acid tethering: In nature, colanic acid is associated with but not attached to the cell surface. To facilitate whole cell encapsulation, we have modified a third pathway to fix the colanic acid to the surface of the cell. This involves the over–production of an enzyme called Rfal.

About Rfal

Module 2 - Encapsulation