IGEM:Imperial/2010/2010/07/20

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Fast Response Module - Key issues - Priority

Quorum sensing mechanism

cross-talking pathways – heterodimerisation (heterodimerising pathways) in (G+)

Protease and fusion protein

TEV protease

  • Structural analysis
  • split mechanism
  • kinetic information

Alternative split/ dimerizing proteases to use.

Alternatives: Phosphate-inducible/ allosteric activating proteases- look at the system, can we manipulate or use them some how?


Two Component system engineering

Synbio applications using EnvZ-OmpR 2CS (what hybrid receptors have been made to what stimuli) EnvZ receptor structure hybrid receptors that have been made and how they were assembled

animation for EnvZ- OmpR – TEV


Alternative pathways:

  • AIP (Autoinducing peptide) induced systems and crosstalk heterodimers.

Screen list of 2CS check for dimerization mechanism, how system is triggered, look for new ones (newcastle igem ref)

Search for gram positive 2CS, get a clear diagram and structural info of receptors if possible (Agr Com)


Checklist for each pathway:

  • Triggering stimulus and receptor
  • Signal transduction pathway
  • Heterodimer formation conformational changes associated with dimerisation (eg OmpR and TEV folding)
  • Protein structure information – receptor, transcription factor
  • What bacterium was the pathway studied in
  • Interaction with other pathways (esp those in B.sub)


OmpR structural analysis- has it been fused?

Contact structural biologist about OmpR-TEV protease fusion


  1. Kinetics
  2. Receptor activation (Env Z etc)
  3. Phosphor-elay
  4. TEV activation / turnover

Specificity of TEV protease and activity of split

Fret pair

Selection and engineer the link between the two – length of sequence – will it still FRET


Colourless to coloured pigments – smth outside fluorescence Smells, etc anything other than fret.


Chassis

  • crosstalk with new pathways (endogenous envZ, ko strains, how/where previous engineering was done)
  • What pathways are present in Bacillus subtilus


List of biobrick parts

We could use- screen previous iGEM teams

2CS QS systems

Detection Module - Key issues

Induction of invasive behaviour

  • Order lipids
  • Can the lipids be made by our chassis?

Specificity of schistosoma proteases

  • Need to be sure that they’ll cut the membrane-anchored protein in the right place and quickly enough (elastase: SWKP)
  • Get hold of the protease and assay it

Could adding the protease recognition site to the AIP effect its properties enough to prevent it from binding the receptor?

Membrane-anchored protein

  • Must make a fusion protein consisting of:
  1. A well-known membrane protein which has been used for fusion proteins before
  2. Either one, or repeating units of our AIP (autoinducing peptide) which, when cleaved, will bind to the receptor
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