Team:Imperial College London/M2

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==Overview==
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<a href="https://static.igem.org/mediawiki/2009/2/2e/II09_MapIndicator_Module2.png" class="highslide" onclick="return hs.expand(this, config1)" title="After autoinduction, the encapsulation and freeze drying genes are expressed.">
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<img src="https://static.igem.org/mediawiki/2009/2/2e/II09_MapIndicator_Module2.png" alt="" title="Click to enlarge" width="75%"/>
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Module 2: Encapsulation
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=[[Image:II09_Thumb_m2.png|40px]]<font size='5'><b>Module 2 - Encapsulsation</b></font>=
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<br>
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<b><i>The E.ncapsulator</i></b> 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.
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<b>WHAT</b>
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<a href="https://static.igem.org/mediawiki/2009/e/e3/Module2Timeline.png" class="highslide" onclick="return hs.expand(this, config1)">
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<img src="https://static.igem.org/mediawiki/2009/e/e3/Module2Timeline.png" alt="" title="Click to enlarge" width="75%"/>
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Module 2 Timeline
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<br>
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Module 2 is a distinct stage that involves the production of an extracellular protective polysaccharide (colanic acid), acid resistance proteins, and storage metabolites.
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==Rationale==
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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.
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<b>WHEN</b>
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/Stomach"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png"></a></html>&nbsp; <b><i>About proteolysis.</i></b>
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Module 2 is initiated following the completion of protein production (Module 1) and prior to genomic neutralisation (Module 3). It should be noted that Module 1 protein production will continue throughout Module 2 but at a heavily reduced rate such that additional resources can be devoted to Module 2 activity.
 
 +
To tackle this seemingly insurmountable problem we adopted a two phase approach.
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<b>WHY</b>
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<b>Phase 1:</b> Identify a suitable chassis with the genotypic potential for acid–resistance.
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Product storage is an important requirement of our system. To achieve this, trehalose is produced during Module 2 to help maintain structural integrety during the freeze drying process. Trehalose additionally, protects the internal 'protein payload' from free radical attack.
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<b>Phase 2:</b>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, <i>Lactobacillus</i>, <i>E.coli</i> and <i>B.subtilis</i> were shortlisted as potential chassis.
 +
Of these three organisms, <i>E.coli</i> was chosen as it is safe, easy to work with and possesses a broad range of acid resistance strategies.
 +
====E.coli and acid resistance:====
 +
[[Image:AcidBath.png|left|200px]] While <i>E.coli</i> has endogenous acid resistance pathways, colonisation of the gut is based on a "numbers approach". In essence, the majority of <i>E.coli</i> 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.
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Module 2 of our system consists of 2 parts: <br>
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This point is illustrated one of our experiments shown by the image on the left. In this experiment, constitutive GFP producing <i>E.coli</i> 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.
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i) An encapsulation mechanism - through which our cells secrete an exopolysaccharide capsule, to protect the cell from the harsh conditions encountered in the stomach.<br>
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ii) A storage mechanism - through which a disaccharide (trehalose) is produced within the cell, with the intention of conferring our protein of interest with some resistance to cold shock and dessication.<br><br>
+
 +
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 <i>E.coli</i>.
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=== Module 2 Part i: Encapsulation ===
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<br><br><br><br><br><br>
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In order to deliver our protein of interest to the gut, we must find some way of conferring our cells with some ability to withstand the harsh conditions present within the stomach. <br><br>
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==Phase 2: Boosting Acid Resistance==
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Escheria coli naturally has a mechanism for withstanding these conditions, allowing them to survive through to the gut. This mechanism consists of secretion of a 'slime capsule,' which allows the cells to resist the highly acidic environment of the stomach. This 'slime capsule' mainly consists of secreted exopolysaccharides, such as colanic acid. By upregulating these genes that are naturally present, this will give our cells the ability to 'auto-encapsulate' - to secrete this slime capsule when required, and allowing our cells to bypass the stomach. <br><br>
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There are several genes required for synthesis of colanic acid.
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RcsB, WaaL, B3023
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This image shows a cross section of our cells, consisting of our protein of interest within, surrounded by ...
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By <i>'hacking' E.coli’s</i> 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.
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[[Image:CAPSULE.JPG]]
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/7/7b/TripleHack.png" class="highslide" onclick="return hs.expand(this, config1)">
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<img src="https://static.igem.org/mediawiki/2009/7/7b/TripleHack.png" alt="" title="Click to enlarge" width="70%"/>
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</a>
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<div class="highslide-caption">
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Module 2: The Triple Hack
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===RcsB===
 
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====RcsB upregulates the following genes:====
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<br>
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=====<b><i>ivy</i> (Inhibitor of Vertebrate lysozyme) </b>=====
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====<b>Acid Resistant Polymer – Colanic acid:</b>====
 +
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] ([http://partsregistry.org/Part:BBa_K200000 BBa_K200000]).
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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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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M2/genes"><img width=50px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a></html>&nbsp;<b><i>About RcsB</i></b>
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<br>
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<br>
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 +
====<b>Safety  – Biofilm prevention:</b>====
 +
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:BBa_K200002 BBa_K200002]).
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<html><a href="https://2009.igem.org/Team:Imperial_College_London/M2/YgiV"><img width=50px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a></html>&nbsp; <b><i>About YgiV</i></b>
 +
<br>
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<br>
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 +
====<b>Microencapsulation – Colanic acid tethering:</b>====
 +
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]).
 +
 
 +
<html><a href="https://2009.igem.org/Team:Imperial_College_London/M2/Rfal"><img width=50px src="http://i691.photobucket.com/albums/vv271/dk806/II09_Learnmore.png" align="left"></a></html>&nbsp; <b><i>About Rfal</i></b>
 +
 
 +
 
 +
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:
 +
 
 +
<b>[http://partsregistry.org/Part:BBa_K200025 BBa_K200025]</b>
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<html>
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<left>
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/b/b0/PcstARcsBTC.png" class="highslide" onclick="return hs.expand(this, config1)">
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<img src="https://static.igem.org/mediawiki/2009/b/b0/PcstARcsBTC.png" alt="" title="Click to enlarge" width="45%"/>
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</a>
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<div class="highslide-caption">
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Module 2: Encapsulation Testing Construct
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</div>
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</left>
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</html>
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<b>[http://partsregistry.org/Part:BBa_K200029 BBa_K200029]</b>
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<html>
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<left>
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/6/61/LacIpLARcsBTC.png" class="highslide" onclick="return hs.expand(this, config1)">
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<img src="https://static.igem.org/mediawiki/2009/6/61/LacIpLARcsBTC.png" alt="" title="Click to enlarge" width="45%"/>
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</a>
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<div class="highslide-caption">
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Module 2: Encapsulation Testing Construct
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</div>
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</div>
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</left>
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</html>
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{{Imperial/09/Division}}
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==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 <i>OtsA</i> and <i>OtsB</i> are required for trehalose production [6].
 +
 
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<html>
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<center>
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/0/0a/TrehaloseModule2Genes.png" class="highslide" onclick="return hs.expand(this, config1)">
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<img src="https://static.igem.org/mediawiki/2009/0/0a/TrehaloseModule2Genes.png" alt="" title="Click to enlarge" width="70%"/>
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</a>
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<div class="highslide-caption">
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Module 2: Initiation
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</div>
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</div>
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</center>
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</html>
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 +
==Freeze Drying Results:==
 +
 
 +
To characterise the protective effect of trehalose against freeze drying, we assembled the following testing constructs:
 +
 
 +
 
 +
<b>[http://partsregistry.org/Part:BBa_K200017 BBa_K200017]</b>
 +
 
 +
<html>
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<left>
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<div class="highslide-gallery">
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<a href="https://static.igem.org/mediawiki/2009/4/45/OtsBTestingConstruct.png" class="highslide" onclick="return hs.expand(this, config1)">
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<img src="https://static.igem.org/mediawiki/2009/4/45/OtsBTestingConstruct.png" alt="" title="Click to enlarge" width="30%"/>
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</a>
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<div class="highslide-caption">
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Module 2: Trehalose Production Testing Construct
 +
</div>
 +
</div>
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</left>
 +
</html>
 +
 
 +
==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] [http://www.ncbi.nlm.nih.gov/pubmed/19139876 Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92]
 +
 
 +
[2] [http://www.ncbi.nlm.nih.gov/pubmed/19026860 Capsular polysaccharides in Escherichia coli.]
 +
 
 +
[3] [http://www.ncbi.nlm.nih.gov/pubmed/11758943 Characterization of the RcsC-->YojN-->RcsB phosphorelay signaling pathway involved in capsular synthesis in Escherichia coli.]
 +
 
 +
[4] [http://www.nature.com/ismej/journal/v2/n6/abs/ismej200824a.html Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)]
 +
 
 +
[5] [http://www.ncbi.nlm.nih.gov/pubmed/19019161 Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase.]
 +
 
 +
[6] [http://www.ncbi.nlm.nih.gov/pubmed/19646542 Resistance of a recombinant Escherichia coli to dehydration.]
-
=====<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>
+
<center>
-
*[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>]
+
===Project Tour===
 +
<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/Autoinduction"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/AIL.jpg"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Thermoinduction"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/TIR.jpg"></a></center>
 +
</html>
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<br>
 +
<hr>
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====RcsB downregulates the following genes:====
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===Module 2 Contents===
 +
<html><table border="0" style="background-color:transparent;" width="60%">
 +
<tr>
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<td width="20%"><a href="https://2009.igem.org/Team:Imperial_College_London/M2/Genetic"><img style="vertical-align:bottom;" width="100%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_geneticcircuit1.png"><br><b>Genetic Circuit</b></a></td>
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*
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Module_2"><img style="vertical-align:bottom;" width="100%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"><br><b>Wet Lab</b></a></center></td>
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=== Module 2 Part ii: Trehalose Production ===
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<td width="20%"><center><a
 +
href="https://2009.igem.org/Team:Imperial_College_London/Drylab"><img style="vertical-align:bottom;" width="100%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage6.png"><br><b>Modelling</b></a></center></td>
 +
</tr></table></html>
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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>
+
{{Imperial/09/TemplateBottom}}
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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>
+
-
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. {{Imperial/09/TemplateBottom}}
+

Latest revision as of 03:55, 22 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] ([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:BBa_K200002 BBa_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]

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


[http://partsregistry.org/Part:BBa_K200017 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] [http://www.ncbi.nlm.nih.gov/pubmed/19139876 Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92]

[2] [http://www.ncbi.nlm.nih.gov/pubmed/19026860 Capsular polysaccharides in Escherichia coli.]

[3] [http://www.ncbi.nlm.nih.gov/pubmed/11758943 Characterization of the RcsC-->YojN-->RcsB phosphorelay signaling pathway involved in capsular synthesis in Escherichia coli.]

[4] [http://www.nature.com/ismej/journal/v2/n6/abs/ismej200824a.html Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)]

[5] [http://www.ncbi.nlm.nih.gov/pubmed/19019161 Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase.]

[6] [http://www.ncbi.nlm.nih.gov/pubmed/19646542 Resistance of a recombinant Escherichia coli to dehydration.]


Project Tour



Module 2 Contents


Genetic Circuit

Wet Lab

Modelling

Mr. Gene   Geneart   Clontech   Giant Microbes