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| | [[Image:II09_TimelineM2.png |center| 600px]] | | [[Image:II09_TimelineM2.png |center| 600px]] |
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| - | ==<b>The need for encapsulation:</b>==
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| - | 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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| - | This point is illustrated by the experiment shown below:
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| - | [[Image:AcidBath.png|left|150px]]
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| - | 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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| - | To tackle this seemingly insurmountable problem we adopted a two phase approach.
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| - | <b>Phase 1:</b> Identify a suitable chassis with the genotypic potential for acid–resistance.
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| - | <b>Phase 2:</b>Manipulate endogenous acid resistance pathways to control the acid resistant phenotype.
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| | {{Imperial/09/Division}} | | {{Imperial/09/Division}} |
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| - | ==<b>Phase 1:</b>==
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| - | 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.
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| - | Based on natural sources of acid resistance, <i>Lactobacillus</i>, <i>E.coli</i> and <i>B.subtilis</i> were shortlisted as potential chassis.
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| - | 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.
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| - | {{Imperial/09/Division}}
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| - | ==<b>Phase 2:</b>==
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| - | We hacked E.coli’s acid resistance module in three places to achieve the production of a safe acid resistant capsule.
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| - | <b>Acid Resistant Polymer – Colanic acid:</b>
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| - | 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.
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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> <b><i>About RcsB</i></b>
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| - | <b>Safety – Biofilm prevention:</b>
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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 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.
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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> <b><i>About YgiV</i></b>
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| - | <b>Microencapsulation – Colanic acid tethering:</b>
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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 the over–production of an enzyme called Rfal.
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| - | <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> <b><i>About Rfal</i></b>
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Module 2 - Acid Resistance Overview
