Team:Edinburgh/Project

From 2011.igem.org

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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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'''This page is obsolete.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 create '''"bioreactors"''', consisting of scaffolds for various enzymes, to carry out reactions in an extracellular environment. The hope is that, by combining the activity of multiple '''synergistic''' enzymes in a small space, high efficiency will be achieved. At least 4 systems are being considered:
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==Designs==
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* As a baseline, use '''bacteria''' as the scaffold, and attach enzymes by cell-surface display techniques.
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The general formats for the basic phage and cell display would be:
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* As a modified version of the above, use bacteria, but concentrate the enzymes on a small part of it such as the '''flagella'''.
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* Promoter-RBS-LeaderSequence-Enzyme-(Linker?)-pVIII
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* Promoter-RBS-INP-(Linker?)-Enzyme
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* As a fairly novel concept, use '''M13 phage''' as the scaffold, and attach enzymes by phage-display techniques to the pVIII coat protein.
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* As a modified version of the above, attach multiple such phage to '''beads''' via the pIII protein, making a larger "reactor".
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As example systems, we will (probably!) use '''cellulases''' as our enzymes of interest.
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Additionally, it would be good if we could also create something from the sugar we hopefully generate. This would involve creation of a '''biorefinery'''.
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==Pages==
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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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==Actions that ought to be carried out==
==Actions that ought to be carried out==
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* Acquire M13 phage.
* Acquire M13 phage.
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* Make or acquire fusion-ready pVIII gene.
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* PCR from our [http://www.neb.com/nebecomm/products/productn4018.asp M13mp18] to get:
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** Remember it has a leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
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** pVIII mature protein
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** Our final fusion will need the leader sequence followed by the gene of interest followed by pVIII proper.
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** pVIII leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
* Make or acquire fusion-ready pIII gene? (optional)
* Make or acquire fusion-ready pIII gene? (optional)
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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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