Team:Hong Kong HKU

From 2013.igem.org

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Project Description:
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Background:
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<b>Bacterial Microcompartments (BMCs)</b> are closed polyhedral macromolecular complexes with a diameter of 100-150 nm, enclosing enzymes and cofactors for various metabolism reactions. One of the example of such endogenous capsid micro-reactors is the <b><u>E</u>thanolamine <u>ut</u>ilization (Eut)</b> BMC from <i>Salmonella enterica</i> spp.
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Bacterial Microcompartments (MCP) are closed polyhedral shells, diameter of 100-150nm, which is made of thin protein sheets, enclosing enzymes and cofactors for various forms of fermentative metabolism. Salmonella enteric enthanolamine ultilization (Eut) MCP is one example.
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In <i>Salmonella enterica</i> spp., 5 genes (Eut S, M, N, L, K) encodes thousands of copies of shell proteins to form a heterogenous MCP shell. Such empty, recombinant Eut BMCs has been successfully cloned and expressed in <i>Escherichia coli</i>. In addition, a localization signal has been identified which can be fused into the N-terminus of unnaturally-encapsulated enzymes or proteins to facilitate their localization into the microcompartment
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5 genes (Eut S, M, N, L, K) encodes thousands of copies of shell proteins to form a heterogenous MCP shell and empty recombinant Eut Shell has been successfully expressed in E.coli.  
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<br><br>
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In addition, a localization signal (N terminal of EutC19) has been identified and signal fused enzymes/ proteins could be targeted to the cavity of the MCP.
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Inspired by these studies, we speculated that this Eut BMC can become a versatile tool if we can:
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<br>(a) modify the exterior surface to confer various novel physical, chemical, and biochemical properties to the whole capsule;
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Inspired by these studies, we think this Eut MCP can become a versatile tool if we can:
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<br>(b) localize special enzyme into the BMC for specfic functionalization;
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<br>(a) Modify its exterior surface to enable surface display
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<br>(c) store or recycle useful and harmful molecules into the BMC
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<br>(b) Localize special enzyme into the MCP to enable specific metabolism
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<br>(c) Store useful or harmful molecules into the MCP
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<!---someone fix the alignment please, also note that the bolding and codings are not Wiki-styled, there must be some fundamental coding problems occuring here--->
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<img src="https://static.igem.org/mediawiki/2013/8/87/BMC_with_His_tag_Fig.1_00000.jpg" width="240" height="216">
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<img src="https://static.igem.org/mediawiki/2013/5/52/PPK1_in_action_design_Fig.2_00000.jpg" width="240" height="216">
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As a proof of concept, we displayed His tag on the surface of the Eut BMCs (Fig. 1), and at the same time, we fused the localization signal to polyphosphate kinase 1 from <i>Tannerella forsythia</i> (Fig. 2). After cloning both of the above constructs into <i>Escherichia coli</i>, we hope to demonstrate the one application of such integration by recycling phosphates from the polluted waters aided by the activity of polyphosphate kinase and encapsulating characteristics of Eut BMC.
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In this iGEM project, using Eut Microcompartment, we aim to show:
 
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<br>(1) MCP shell surface can be used to display specific peptides, without disrupting the MCP structure, to enable cell-specific targeting for drug delivery system, easy purification etc. We will display Flag and His tag as a proof of concept.
 
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<br>(2) Localize polyphosphate kinase (ppk1) into the native/ engineered MCP to polymerize inorganic phosphate and accumulate them in MCP, trying to treat phosphate pollution in waste water.
 
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We will divide into 2 parallel sub-teams, specifically working on surface decoration and ppk enzyme.
 
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Revision as of 16:50, 23 September 2013




Bacterial Microcompartments (BMCs) are closed polyhedral macromolecular complexes with a diameter of 100-150 nm, enclosing enzymes and cofactors for various metabolism reactions. One of the example of such endogenous capsid micro-reactors is the Ethanolamine utilization (Eut) BMC from Salmonella enterica spp.

In Salmonella enterica spp., 5 genes (Eut S, M, N, L, K) encodes thousands of copies of shell proteins to form a heterogenous MCP shell. Such empty, recombinant Eut BMCs has been successfully cloned and expressed in Escherichia coli. In addition, a localization signal has been identified which can be fused into the N-terminus of unnaturally-encapsulated enzymes or proteins to facilitate their localization into the microcompartment

Inspired by these studies, we speculated that this Eut BMC can become a versatile tool if we can:
(a) modify the exterior surface to confer various novel physical, chemical, and biochemical properties to the whole capsule;
(b) localize special enzyme into the BMC for specfic functionalization;
(c) store or recycle useful and harmful molecules into the BMC



As a proof of concept, we displayed His tag on the surface of the Eut BMCs (Fig. 1), and at the same time, we fused the localization signal to polyphosphate kinase 1 from Tannerella forsythia (Fig. 2). After cloning both of the above constructs into Escherichia coli, we hope to demonstrate the one application of such integration by recycling phosphates from the polluted waters aided by the activity of polyphosphate kinase and encapsulating characteristics of Eut BMC.

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