IGEM:Imperial/2010/Strategy

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Current revision (06:01, 27 September 2010) (view source)
(Strategy: 27th Sept update)
 
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{{Imperial2010/Header}}
=Strategy=  
=Strategy=  
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==Assembly strategy and Lab-work==
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Please refer to the assembly strategy for details.  
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Please refer to the overall assembly strategy for details.  
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Three Lab-teams are currently working in parallel to assemble parts which will be needed for down-stream steps, as soon as the synthesized sequences arrive.  
Three Lab-teams are currently working in parallel to assemble parts which will be needed for down-stream steps, as soon as the synthesized sequences arrive.  
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LINKS to lab teams.
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*[[IGEM:Imperial/2010/XylE_team | XylE team (Maddie and Nicolas)]]
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*[[IGEM:Imperial/2010/Vectors_team | Vectors team (Kyasha and Kirill)]]
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*[[IGEM:Imperial/2010/Surface_Protein_team | Surface Protein team (Harriet and Florian)]]
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'''Key updates:'''
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* ''XylE characterization'': (27th Sept) Assays have been started. First data provides important clues on required catechol concentrations, response time and absorbance of the catechol-breakdown product. Assays for XylE responses will now be based on absorbance at 380nm. An assay  attempting to determine deleteriousness of the catechol XylE assay for cells has failed to produce consistent data and will be repeated. The LacI-controlled XylE will is being assembled and will allow in-vivo testing. The sumo-XylE construct is about to be finished (in-vitro testing).
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* ''GFP-XylE characterization'': (27th Sept) Cloning of PveG with optimized RBS has not been successful so far. This prevents us from proceeding into the in-vitro testing phase, since the finished GFP-XylE fusion cannot be expressed.
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* ''PryD-vector'': (27th Sept) assembly is finished.
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* ''AmyE-vector'': (27th Sept) full assembly is still prevented by difficulties with Dif-PME transformations. Progress is expected during the week as Kirstin has been asked for assistance.
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* ''Surface protein'': (27th Sept) assembly has not proceeded to a satisfying state. Severe problems with digestions and transformations of LytC and the pveg-promoter have been delaying progress. However the previously assembled terminator vector has already been successfully used in several other assembly lines.
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* Synthesis: (27th Sept) We are still waiting for the arrival of synthesised ComE and two Linker constructs. As the sequences are not required just yet, they are currently not slowing down our progress. LacI-TEV and ComD have arrived.  
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==Assembly strategy==
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LINKS to shedule history (?)
 
[[Image:Assembly strat 1.1 Pic.JPG|Center|1000px|x600px| Assembly strategy version 1.1]]
[[Image:Assembly strat 1.1 Pic.JPG|Center|1000px|x600px| Assembly strategy version 1.1]]
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[[Image:PrimersPic1.bmp|thumb|right|600px|x400px| '''Additional primers - work in progress''']]
[[Image:PrimersPic1.bmp|thumb|right|600px|x400px| '''Additional primers - work in progress''']]
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* Urgent primers have been ordered on Monday 16th.
 
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* 14 other primers will be required. Design is in progress (pic of word table)!
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* 14 other primers will be required. Design is in progress (pic of word tabl)- have been ordered (OK)
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* Experimental design for XylE e.coli testing!
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* Experimental design for XylE e.coli testing! (OK)
* Functional testing strategies have been developed and have been indicated in the overall assembly strategy diagram. Need to be written up properly!
* Functional testing strategies have been developed and have been indicated in the overall assembly strategy diagram. Need to be written up properly!
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* XylE and TEV will not be tagged. In order to be able to analyse their activity, we will compare substrate-turnover rates.
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* Strategies for testing of expression etc have to be developed and structured. (OK)
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* Strategies for testing of expression etc have to be developed and structured.
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* List of parts for submission has to be assembled.  
* List of parts for submission has to be assembled.  
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* Gant chart!
 
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* How much synthetic AIP will be required to trigger our test system? (Modellers)
 
* ComE promoter system - recap on mode of function.
* ComE promoter system - recap on mode of function.
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[[Image:Primers1.1.docx]]

Current revision

Strategy

Please refer to the assembly strategy for details.

Three Lab-teams are currently working in parallel to assemble parts which will be needed for down-stream steps, as soon as the synthesized sequences arrive.

Key updates:

  • XylE characterization: (27th Sept) Assays have been started. First data provides important clues on required catechol concentrations, response time and absorbance of the catechol-breakdown product. Assays for XylE responses will now be based on absorbance at 380nm. An assay attempting to determine deleteriousness of the catechol XylE assay for cells has failed to produce consistent data and will be repeated. The LacI-controlled XylE will is being assembled and will allow in-vivo testing. The sumo-XylE construct is about to be finished (in-vitro testing).
  • GFP-XylE characterization: (27th Sept) Cloning of PveG with optimized RBS has not been successful so far. This prevents us from proceeding into the in-vitro testing phase, since the finished GFP-XylE fusion cannot be expressed.
  • PryD-vector: (27th Sept) assembly is finished.
  • AmyE-vector: (27th Sept) full assembly is still prevented by difficulties with Dif-PME transformations. Progress is expected during the week as Kirstin has been asked for assistance.
  • Surface protein: (27th Sept) assembly has not proceeded to a satisfying state. Severe problems with digestions and transformations of LytC and the pveg-promoter have been delaying progress. However the previously assembled terminator vector has already been successfully used in several other assembly lines.
  • Synthesis: (27th Sept) We are still waiting for the arrival of synthesised ComE and two Linker constructs. As the sequences are not required just yet, they are currently not slowing down our progress. LacI-TEV and ComD have arrived.



Assembly strategy

Assembly strategy version 1.1

Wolfs checklist

Additional primers - work in progress
Additional primers - work in progress
  • 14 other primers will be required. Design is in progress (pic of word tabl)- have been ordered (OK)
  • Experimental design for XylE e.coli testing! (OK)
  • Functional testing strategies have been developed and have been indicated in the overall assembly strategy diagram. Need to be written up properly!
  • Strategies for testing of expression etc have to be developed and structured. (OK)
  • List of parts for submission has to be assembled.
  • ComE promoter system - recap on mode of function.

Image:Primers1.1.docx

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