Team:Imperial College London/M2

   Module 2: Encapsulation =Module 2 - Encapsulsation =

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:
While E.coli has endogenous acid resistance pathways, colonisation of the gut is based on a "numbers approach". In essence, the majority of E.coli cells in a population do not survive passage through the stomach but the few that do are able to regenerate the population once in the intestine.

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 RcsB [3] (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] (BBa_K200002). <img width=50px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a> 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] (BBa_K200003).

<img width=50px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a> 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:

BBa_K200025

 <img src="http://2009.igem.org/wiki/images/b/b0/PcstARcsBTC.png" alt="" title="Click to enlarge" width="45%"/> </a> Module 2: Encapsulation Testing Construct

BBa_K200029

 <img src="http://2009.igem.org/wiki/images/6/61/LacIpLARcsBTC.png" alt="" title="Click to enlarge" width="45%"/> </a> Module 2: Encapsulation Testing Construct

Freeze Drying Theory:
Freeze drying is the process by which a material is preserved via dehydration at a very low temperature. Freeze drying our chassis would slow the decomposition of the polypeptide and prevent the growth of any contaminanting micro-organisms. Unfortunatly, freeze drying can seriously damage cell membranes and denature the internal polypeptides. To prevent this from occuring we have decided to upregulate the biosynthesis of the cryoprotectant trehalose. 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 E.coli. The genes OtsA and OtsB are required for trehalose production [6].

 <img src="http://2009.igem.org/wiki/images/0/0a/TrehaloseModule2Genes.png" alt="" title="Click to enlarge" width="70%"/> </a> Module 2: Initiation

Freeze Drying Results:
To characterise the protective effect of trehalose against freeze drying, we assembled the following testing constructs:

BBa_K200017

 <img src="http://2009.igem.org/wiki/images/4/45/OtsBTestingConstruct.png" alt="" title="Click to enlarge" width="30%"/> </a> Module 2: Trehalose Production Testing Construct

Conclusion:
We have selected E.coli as the most suitable chassis for encapsulation and carefully modulated endogenous pathways to result in the synthesis of a safe colanic acid capsule. In addition, we have designed a treahlose production system that will faciliate the storage of our final product for extended periods of time.

References:
[1] Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92

[2] Capsular polysaccharides in Escherichia coli.

[3] Characterization of the RcsC-->YojN-->RcsB phosphorelay signaling pathway involved in capsular synthesis in Escherichia coli.

[4] Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

[5] Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase.

[6] Resistance of a recombinant Escherichia coli to dehydration.

Project Tour
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