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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'''This page is obsolete.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 "reactors", 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 two different systems will be investigated:
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==Designs==
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* Using M13 phage as the scaffold, and attaching enzymes by cell-surface display techniques to the pVIII coat protein.
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The general formats for the basic phage and cell display would be:
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** Additionally, it may be possible to attach multiple phage to beads, making a larger "reactor".
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* Using bacteria as the scaffold, and attaching enzymes by cell-surface display techniques.
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** This ought to be less efficient, but it may be possible to localise enzymes to the pili or flagella, increasing the synergy...
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As an example system, we will try 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.
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* Promoter-RBS-LeaderSequence-Enzyme-(Linker?)-pVIII
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* Promoter-RBS-INP-(Linker?)-Enzyme
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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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==Actions that ought to be carried out==
==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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* Acquire M13 phage.
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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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* If a simple test system is desired, make fusion-ready amylase? Or something else?
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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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* Make or acquire fusion-ready pVIII gene.
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===Phage display===
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** Perhaps from <partinfo>BBa_K415151</partinfo>.
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** Remember it has a leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
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* Acquire M13 phage.
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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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* 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.
* Make or acquire fusion-ready pIII gene? (optional)
* Make or acquire fusion-ready pIII gene? (optional)
** See <partinfo>BBa_K415138</partinfo> (complete) or <partinfo>BBa_K257001</partinfo> (''sans'' leader sequence).
** 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.
** Our final fusion will need the leader sequence followed by the gene of interest followed by pIII proper.
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===Cell display===
* Make or acquire fusion-ready cell-surface display parts.
* 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].
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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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** Maybe also see <partinfo>BBa_K265008</partinfo>.
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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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* Make or acquire spacer/linker sequences?
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===Biorefinery===
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** These are short enough that ordering them as oligos probably makes sense...
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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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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!


Retrieved from "http://2011.igem.org/Team:Edinburgh/Project"