Module 2 - Encapsulsation

Module 2: Encapsulation

The E.ncapsulator has been designed to be able to withstand the rigours of stomach acid and freeze drying. This is achieved by the synthesis of the exopolysaccharide colanic acid and the cryoprotectant trehalose.

Module 2 Timeline

Rationale

To ferry polypeptides through the stomach, each of our microcapsules must withstand a heated protease–rich acidic environment in the stomach. Few chassis have the potential to withstand this harsh environment. Since our microcapsules are inanimate, we cannot rely on any of the active acid–resistance strategies that living bacteria are able to deploy.

  About proteolysis.

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: Which Chassis?

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.

Of these three organisms, E.coli was chosen as it is safe, easy to work with and possesses a broad range of acid resistance strategies.

E.coli and acid resistance:

This point is illustrated one of our experiments shown by the image on the left. In this experiment, constitutive GFP producing E.coli cells were exposed to differing concentrations of acid for 30 minutes. The reduction in fluoresence is indicative of cell lysis and the subsequent acid-induced denaturing of GFP.

This experiment indicates that if we are to deliver a significant dose of a given polypeptide therapeutic past the stomach, it will be necessary to boost the natural acid resistance of E.coli.

Phase 2: Boosting Acid Resistance

By 'hacking' E.coli’s endogenous acid resistance pathways in three places we induced the formation of a safe colanic acid microcapsule that protects against the harsh acidic conditions of the stomach ¹. Without encapsulation, our polypeptides would be denatured and degraded by stomach acid and digestive proteases respectively.

Module 2: The Triple Hack

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 ². 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 a gene called RcsB³ ([http://partsregistry.org/Part:BBa_K200000 BBa_K200000]).

 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 ⁴ ([http://partsregistry.org/Part:K200002 K200002]).

  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 ⁵ ([http://partsregistry.org/Part:BBa_K200003 BBa_K200003]).

  About Rfal

Based on the literature, the natural formation of a colanic acid capsule naturally takes about 2-3 days. We hope that the upregulation of RcsB will shorten this time. Either way, we will be able to share more of our results with you at the Jamboree.

Acid Resistance Results:

To characterise colanic acid encapsulation, we assembled the following testing constructs:

[http://partsregistry.org/Part:BBa_K200025 BBa_K200025]

Module 2: Encapsulation Testing Construct

[http://partsregistry.org/Part:BBa_K200029 BBa_K200029]

Module 2: Encapsulation Testing Construct

Module 2 Contents