Team:Edinburgh/Project

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A [[Project descriptions|project description]] and [[Safety/Proposals|safety proposal]] is supposed to be submitted by July 15.
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__NOTOC__
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'''This page is obsolete. The project should be documented at the relevant subpages:'''
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==Project abstract==
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* [[Team:Edinburgh/Phage Reactors 2.0|Phage Reactors]]
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* [[Team:Edinburgh/Cell Display|Cell Display]]
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* [[Team:Edinburgh/Biorefineries|Biorefineries]]
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This year we will investigate enzyme synergy outside the cell, by creating ''E. coli'' with different cellulase enzymes displayed on the surface, as well as (if possible) M13 phage with the same enzymes displayed on the pVIII coat protein (or pIII might be used as a fallback).
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==Designs==
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==Pages==
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The general formats for the basic phage and cell display would be:
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* [[Team:Edinburgh/Phage Reactors 2.0|Phage Reactors]]
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* Promoter-RBS-LeaderSequence-Enzyme-(Linker?)-pVIII
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* [[Team:Edinburgh/Cell Display|Cell Display]]
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* Promoter-RBS-INP-(Linker?)-Enzyme
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==Todo==
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==Actions that ought to be carried out==
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===General===
* Make or acquire fusion-ready cellulases (e.g. <partinfo>BBa_K392006</partinfo>, <partinfo>BBa_K392007</partinfo>, <partinfo>BBa_K392008</partinfo>).
* Make or acquire fusion-ready cellulases (e.g. <partinfo>BBa_K392006</partinfo>, <partinfo>BBa_K392007</partinfo>, <partinfo>BBa_K392008</partinfo>).
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* Since a simple test system is desired, make fusion-ready amylase, from ''E. coli's'' own ''malS'' gene.
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* Make or acquire spacer/linker sequences?
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** Short linkers could be ordered as oligos. [[Team:Edinburgh/Berkeley 2009 Parts|Berkeley 2009 made some longer linkers]] which we could order.
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===Phage display===
* Acquire M13 phage.
* Acquire M13 phage.
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* If a simple test system is desired, make fusion-ready amylase?
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* PCR from our [http://www.neb.com/nebecomm/products/productn4018.asp M13mp18] to get:
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** pVIII mature protein
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** pVIII leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
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* Make or acquire fusion-ready pVIII gene (perhaps from <partinfo>BBa_K415151</partinfo>).
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* Make or acquire fusion-ready pIII gene? (optional)
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** Remember it has a leader sequence. Our final fusion will need the leader sequence followed by the gene of interest followed by pVIII proper.
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** See <partinfo>BBa_K415138</partinfo> (complete) or <partinfo>BBa_K257001</partinfo> (''sans'' leader sequence).
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** Our final fusion will need the leader sequence followed by the gene of interest followed by pIII proper.
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* Make or acquire fusion-ready pIII gene?
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===Cell display===
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** Remember it has a leader sequence. Our final fusion will need the leader sequence followed by the gene of interest followed by pIII proper.
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* Make or acquire fusion-ready cell-surface display parts (see [[Team:Berkeley Wetlab/Cell Surface Display Parts|Berkeley 2009]]).
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* Make or acquire fusion-ready cell-surface display parts.
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** See [https://2009.igem.org/Team:Berkeley_Wetlab/Cell_Surface_Display_Parts Berkeley 2009] (but perhaps ignore them).
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** Instead, <partinfo>BBa_K265008</partinfo> (ice nucleation protein) looks promising.
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** The ''fliC'' gene seems to be the target for flagella display; note that the displayed protein has to be inserted into the middle of it.
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===Biorefinery===
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:''To add...''
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===Completion===
* Fuse whatever enzymes to cell-display parts or pVIII or maybe pIII.
* Fuse whatever enzymes to cell-display parts or pVIII or maybe pIII.
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* ???
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* '''Profit!'''
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Latest revision as of 14:41, 28 July 2011

This page is obsolete. The project should be documented at the relevant subpages:

Designs

The general formats for the basic phage and cell display would be:

  • Promoter-RBS-LeaderSequence-Enzyme-(Linker?)-pVIII
  • Promoter-RBS-INP-(Linker?)-Enzyme

Actions that ought to be carried out

General

  • Make or acquire fusion-ready cellulases (e.g. <partinfo>BBa_K392006</partinfo>, <partinfo>BBa_K392007</partinfo>, <partinfo>BBa_K392008</partinfo>).
  • Since a simple test system is desired, make fusion-ready amylase, from E. coli's own malS gene.

Phage display

  • Acquire M13 phage.
  • PCR from our [http://www.neb.com/nebecomm/products/productn4018.asp M13mp18] to get:
    • pVIII mature protein
    • pVIII leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
  • Make or acquire fusion-ready pIII gene? (optional)
    • See <partinfo>BBa_K415138</partinfo> (complete) or <partinfo>BBa_K257001</partinfo> (sans leader sequence).
    • Our final fusion will need the leader sequence followed by the gene of interest followed by pIII proper.

Cell display

  • Make or acquire fusion-ready cell-surface display parts.
    • See Berkeley 2009 (but perhaps ignore them).
    • Instead, <partinfo>BBa_K265008</partinfo> (ice nucleation protein) looks promising.
    • The fliC gene seems to be the target for flagella display; note that the displayed protein has to be inserted into the middle of it.

Biorefinery

To add...

Completion

  • Fuse whatever enzymes to cell-display parts or pVIII or maybe pIII.
  •  ???
  • Profit!


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