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'''This page is obsolete.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 '''"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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==Actions that ought to be carried out==
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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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===General===
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As example systems, we will (probably!) use '''cellulases''' as our enzymes of interest.
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* Make or acquire fusion-ready cellulases (e.g. <partinfo>BBa_K392006</partinfo>, <partinfo>BBa_K392007</partinfo>, <partinfo>BBa_K392008</partinfo>).
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==Pages==
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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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* [[Team:Edinburgh/Phage Reactors 2.0|Phage Reactors]]
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* Make or acquire spacer/linker sequences?
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* [[Team:Edinburgh/Cell Display|Cell Display]]
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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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==Actions that ought to be carried out==
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===Phage display===
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* 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.
* Acquire M13 phage.
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* If a simple test system is desired, make fusion-ready amylase? Or something else?
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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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* Make or acquire fusion-ready pVIII gene.
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** pVIII leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
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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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** Our final fusion will need the leader sequence followed by the gene of interest followed by pVIII proper.
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* 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.
** 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.
** 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!


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