IGEM:Imperial/2010/2010/07/20: Difference between revisions
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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 | |||
# Kinetics | |||
# Receptor activation (Env Z etc) | |||
# Phosphor-elay | |||
# 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: | |||
# A well-known membrane protein which has been used for fusion proteins before | |||
# Either one, or repeating units of our AIP (autoinducing peptide) which, when cleaved, will bind to the receptor | |||
Revision as of 07:04, 20 July 2010
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Fast Response Module - Key issues - PriorityQuorum sensing mechanismcross-talking pathways – heterodimerisation (heterodimerising pathways) in (G+) Protease and fusion proteinTEV protease
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 engineeringSynbio 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
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)
Contact structural biologist about OmpR-TEV protease fusion
Specificity of TEV protease and activity of split Fret pairSelection and engineer the link between the two – length of sequence – will it still FRET
Chassis
List of biobrick partsWe could use- screen previous iGEM teams 2CS QS systems Detection Module - Key issuesInduction of invasive behaviour
Specificity of schistosoma proteases
Could adding the protease recognition site to the AIP effect its properties enough to prevent it from binding the receptor? Membrane-anchored protein
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