IGEM:Imperial/2010/Strategy: Difference between revisions
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'''Key updates:''' | '''Key updates:''' | ||
* ''XylE characterization'': | * ''XylE characterization'': Transformation of XylE into the terminator vector has been successful. Assays can be started with. | ||
* ''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. | |||
* 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. | |||
* 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. | |||
Revision as of 03:56, 10 September 2010
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.
- XylE team (Maddie and Nicolas)
- Vectors team (Kyasha and Kirill)
- Surface Protein team (Harriet and Florian)
Key updates:
- XylE characterization: Transformation of XylE into the terminator vector has been successful. Assays can be started with.
- 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.
- 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.
- 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.
Assembly strategy
Wolfs checklist
- 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.
