< IGEM:Imperial/2010(Difference between revisions)
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* ''XylE characterization'':
* ''XylE characterization'': () of XylE . of XylE -will be in-vitro .
of the GFP-XylE fusion
proteinwith and -in .
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.
- XylE team (Maddie and Nicolas)
- Vectors team (Kyasha and Kirill)
- Surface Protein team (Harriet and Florian)
- 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.
- 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.