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{{Imperial2010/Header}}
=Strategy=  
=Strategy=  


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'''Key updates:'''
'''Key updates:'''


* ''XylE characterization'': Transformation of XylE into the terminator vector has been successful. Assays can be started with.  
* ''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'': The GFP-XylE construct has produced some difficulties: after a new GFP primer had to be ordered to create a corrected TEV overhang, the first annealing reactions may have been successful - however confusion in the lab caused loss of the reactions. Currently, Kirstin is working on the annealing reaction and will also PCR-blunt the product into a vector which allows efficient digestion by XbaI and SpeI. Piotr and Nick have been working on the promoter-RBS part necessary for expression of GFP-XylE and are now about to transform. We hope to be able to purify expressed GFP-XylE from e.coli by the end of next week.  


* The ''PryD-vector'' assembly is about to be finished.
* ''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.  
* The ''AmyE-vector'' assembly has been slowed down by excessive background in the transformation of the Oligo (Dif-PmE). A new attempt is currently being carried out and looks promising so far. In case of success,the final step in the AmyE assembly can be carried out.  


* The ''surface protein'' 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.  
* ''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.  


* We are still waiting for the arrival of synthesised LacI-TEV, ComE, ComD and two Linker constructs. As the sequences are not required just yet, they are currently not slowing down our progress.
* ''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.  




Latest revision as of 04:01, 27 September 2010

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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
  • 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.

File:Primers1.1.docx

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