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Team:Imperial College London/M2
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Module 2 feedback from todays session:
2) More detail and add references. Keep it technical so understandable to all.
6) Results: Need pics of plates - CHARLES TO BRING IN CAMERA! Growth assay - tomorrow?!
7) Trehalose - need this but don't have data for freeze drying...
10) Link all constructs to registry.
11) If no data exists - say that as the wiki is being frozen we haven't added the data but will have it in time for the Jamboree.
13) Add what teams can reuse from this module.
Contents |
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.
Rationale:
To ferry polypeptides through the stomach, each of our microcapsules must withstand 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.
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. Without encapsulation, our polypeptides would be denatured and degraded by stomach acid and digestive proteases respectively.
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 a 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
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:
Freeze Drying Theory:
Freeze drying is the process by which a material is preserved via its dehydration at a very low temperature. Freeze drying our chassis would slow the decomposition of the polypeptide therapeutic 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.
Freeze Drying Results:
To characterise the protective effect of trehalose against freeze drying, we assembled the following testing constructs:
Unfortunatly, we did not have time to ligate together both OtsA and OtsB and were therefore unable to do any functional assays.
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:
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