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From 2011.igem.org

A project description and safety proposal is supposed to be submitted by July 15.

Contents 1 Project abstract 2 Pages 3 Actions that ought to be carried out 3.1 General 3.2 Phage display 3.3 Cell display 3.4 Biorefinery 3.5 Completion

Project abstract

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:

• As a baseline, use bacteria as the scaffold, and attach enzymes by cell-surface display techniques.

• As a modified version of the above, use bacteria, but concentrate the enzymes on a small part of it such as the flagella.

• As a fairly novel concept, use M13 phage as the scaffold, and attach enzymes by phage-display techniques to the pVIII coat protein.

• As a modified version of the above, attach multiple such phage to beads via the pIII protein, making a larger "reactor".

As example systems, we will (probably!) use cellulases as our enzymes of interest.

Additionally, it would be good if we could also create something from the sugar we hopefully generate. This would involve creation of a biorefinery.

Pages

• Phage Reactors
• Cell Display
• Biorefineries

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.

• Make or acquire fusion-ready pVIII gene.
  • Remember it has a leader sequence, coding for: MKKSLVLKASVAVATLVPMLSFA.
  • Our final fusion will need the leader sequence followed by the gene of interest followed by pVIII proper.

• 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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