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Team:Edinburgh/Project
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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This page is obsolete.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.
- Make or acquire spacer/linker sequences?
- Short linkers could be ordered as oligos. Berkeley 2009 made some longer linkers which we could order.
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!
