Team:Imperial College London/M2

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===Module 2 Contents===
===Module 2 Contents===
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/M2/Genetic"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_geneticcircuit1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Module_2"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"></a><html><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage6.png"></a><center></html>
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</a><a href="https://2009.igem.org/Team:Imperial_College_London/M2/Genetic"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_geneticcircuit1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Module_2"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"></a><html><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage6.png"></a><center></html>
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=Encapsulation Rationale=
 
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In nature, encapsulation pathways such as spore formation, aliginate biosynthesis and colanic acid production all share one common feature: they require a large number of genes. For this reason, we decided that the best way encapsulate our chassis was via the modulation of an existing pathway.
 
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<i>E.coli</i> naturally produces a harmless acid resistant polymer known as colanic acid. By tapping into the pathway that initiates colanic acid biosynthesis, we can turn on its production via the modulation of a gene called RcsB.
 
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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 the production of a number of virulence factors. To prevent biofilm formation from occuring, 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 called YgiV.
 
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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 an enzyme called Rfal.
 
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M2/genes"><img width=90px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a></html><br><br> &nbsp; About RcsB, YgiV and Rfal.
 
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=Freeze Drying=
 
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[[Image:IceBac2.png|left|250px]]
 
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Cells can be stored for extended periods of time by dehydration. However, under such conditions the integrety of both a cell's membrane and intracellular polypeptides can be compromised.
 
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Trehalose is a disaccharide formed from two glucose molecules that provides resistance to dessication. While trehalose is naturally produced in <i>E.coli</i>, we hope that by upregulating its production, we can confer additional resistance to freeze drying. This would allow easy transport and storage of the final product.
 
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M2/Trehalose"><img width=90px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a></html><br><br> &nbsp; About the protective effects of trehalose.
 
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=Secondary Encapsulation=
 
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We have developed a manufacturing process that enables the assembly of a secondary capsule around the colanic acid coating. This secondary capsule has the purpose of holding the cells together in a pill-like shape.
 
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We have investigated the feasibility several secondary encapsulation technologies.
 
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<b>Milk Protein: (Protein) </b>
 
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<b>Gelatin: (Protein) </b>
 
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<b>Xantham Gum: (Polysaccharide) </b>
 
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For more detail click [[Imperial_College_London/M2/Detail|Here]]
 
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==What:==
 
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<b>Module 2</b> is the encapsulation phase. The cell secretes an extracellular protective polysaccharide (colanic acid) which surrounds the cell.  This forms a protective capsule that can withstand the acidic environment of the stomach. The cells also produce acid resistance proteins and storage metabolites (trehalose) which results in protein shielding and storage respectively.
 
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==Why:==
 
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Colanic acid encapsulation and the synthesis of various acid resistance proteins protect the protein of interest from the digestive assaults of the buccal cavity and acid-filled stomach. Once the pill reaches the intestine, gut microflora will digest the <b><i>E.ncapsulator's</i></b> colanic acid coat, releasing the protein of interest.
 
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Trehalose preserves our protein of interest in its correct conformation in response to dessication. Thus trehalose allows for the freeze drying of the pill, and this will allow easy transport and storage.
 
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==When:==
 
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Encapsulation (<b>Module 2</b>) is initiated following the completion of protein production (<b>Module 1</b>). It should be noted that <b>Module 1</b> protein production continues at a lower 'maintenance level' throughout <b>Module 2</b>.
 
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==How:==
 
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Through the course of evolution, <i>E.coli</i> have equipped themselves with a multitude of defences to enable colanisation of the intestine. We are using two global transcription factors (RcsB & YgiV) to hijack this natural process in a way that maximises acid resitance. We have additionally upregulated a third enzyme (rfal) to enhance the encapsulation of single cells (over and above colony encapsulation). Finally, the two biosynthetic genes (OtsA & OtsB) code for the production of trehalose. Our manipulation of endogenous pathways reduces virulence while enchancing pill functionality.
 
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[[Image:Mod2.jpg| 70px]]
 
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=== Module 2 Part i: Encapsulation ===
 
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===RcsB===
 
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<b>Background:</b>
 
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RcsB is a transcription factor that forms part of the phosphorelay system. In response to membrane stress, RcsB is phosphorylated into its DNA binding form. In this state, it is able to both upregulate and downregulate a large number of genes.
 
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====RcsB upregulates the following genes:====
 
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=====<b><i>ivy</i> (Inhibitor of Vertebrate lysozyme) </b>=====
 
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*Discovered in 2001 as the first bacterial lysozyme inhibitor. This Type-C lysozyme inhibitor resides in the periplasm.<cite>1</cite>
 
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=====<b><i>MilC</i> (Membrane-bound lysozyme inhibitor of Type C lysozyme) </b>=====
 
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*This is a lipoprotein that resides in the membrane. <sup>1</sup>
 
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<b>References</b>
 
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*[http://www.ncbi.nlm.nih.gov/pubmed/19136591 <b>The Rcs two-component system regulates expression of lysozyme inhibitors and is induced by exposure to lysozyme</b>]
 
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====RcsB downregulates the following genes:====
 
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===YgiV===
 
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<b>Background:</b>
 
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In nature, the colanic acid synthesis phase occurs prior to biofilm formation. The latter process of biofilm formation is associated with the upregulation of a number of virulence factors. The transcription factor YgiV blocks the progression into biofilm formation by maintaining colanic acid production. Thus YgiV serves to increase acid resistance and decrease virulence.
 
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===Rfal===
 
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<b>Background:</b>
 
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In the majority of E.coli, the enzyme Rfal joins the O-antigen to the membrane-bound lipid core molecule. Since the K-12 strain has an insertion mutation in the gene coding for O-antigen, the enzyme Rfal is free to join colanic acid to the lipid core.
 
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=== Module 2 Part ii: Trehalose Production ===
 
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An important consideration when designing the specifications of the E.ncapsulator was the ability to store the cells for extended periods of time. This could be achieved by dehydrating the cells. However, normally under such conditions there poses a problem to maintaining the integrity of the proteins within the cells. This is problematic for us, as this could lead to breakdown of our protein of interest. <br><br>
 
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In order to preserve the integrity of our protein of interest during storage of the E.ncapsulator, we decided to incorporate a device for trehalose production within our system. 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 Escherichia Coli. We hope that by upregulating the trehalose production pathways in E.coli we can increase trehalose concentrations within our cell, thereby conferring some resistance to protein degredation in our system. This would allow easy transport and storage of the final product.<br><br>
 
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The trehalose coding region in E.coli consists of 2 genes, OtsA and OtsB - each coding for a different enzyme required for the conversion of glucose to trehalose.
 
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Revision as of 22:15, 21 October 2009

Contents

II09 Thumb m2.pngModule 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 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:

AcidBath.png
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.



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] (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] (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:

BBa_K200025

BBa_K200029


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].

Freeze Drying Results:

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


BBa_K200017

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


Module 2 Contents

Genetic Circuit
Wet Lab
Modelling

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