IGEM:Imperial/2010/Strategy - Revision history

Wolfgang Pernice: /* Strategy */ 27th Sept update

2010-09-27T11:01:05Z

Strategy: 27th Sept update

←Older revision		Revision as of 11:01, 27 September 2010
Line 12:		Line 12:
	'''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.

Benjamin Miller at 16:14, 23 September 2010

2010-09-23T16:14:13Z

←Older revision		Revision as of 16:14, 23 September 2010
Line 1:		Line 1:
		+	{{Imperial2010/Header}}
	=Strategy=		=Strategy=

Wolfgang Pernice: /* Strategy */

2010-09-10T10:56:10Z

Strategy

←Older revision		Revision as of 10:56, 10 September 2010
Line 11:		Line 11:
	'''Key updates:'''		'''Key updates:'''

-	* ''XylE characterization'': ~~During the next week (week 9) we will start characterization~~ of XylE. ~~Purification of XylE~~-~~His tagged from Jeremy~~'~~s cultures will~~ be a first ~~step~~ in ~~direction~~ of in-~~vitro assays~~. ~~Upon assembly of~~ the GFP-XylE ~~fusion protein this~~ week, protein ~~purification will provide us~~ with ~~both XylE~~ and ~~GFP~~-~~XylE for~~ in ~~vitro testing~~. ~~Important kinetic data will hopefully be obtained during~~ the ~~next 7 days~~.	+	* ''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.
		+

Wolfgang Pernice: /* Wolfs checklist */

2010-09-10T10:44:44Z

Wolfs checklist

←Older revision		Revision as of 10:44, 10 September 2010
Line 23:		Line 23:

	[[Image:PrimersPic1.bmp\|thumb\|right\|600px\|x400px\| '''Additional primers - work in progress''']]		[[Image:PrimersPic1.bmp\|thumb\|right\|600px\|x400px\| '''Additional primers - work in progress''']]
-	* Urgent primers have been ordered on Monday 16th.

-	* 14 other primers will be required. Design is in progress (pic of word ~~table~~)!	+	* 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!	+	* 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!

-	* XylE and TEV will not be tagged. In order to be able to analyse their activity, we will compare substrate-turnover rates.	+	* Strategies for testing of expression etc have to be developed and structured. (OK)
-		+
-	* Strategies for testing of expression etc have to be developed and structured.	+

	* List of parts for submission has to be assembled.		* List of parts for submission has to be assembled.
-
-	* Gant chart!
-
-	* 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.

	[[Image:Primers1.1.docx]]		[[Image:Primers1.1.docx]]

Wolfgang Pernice at 11:28, 31 August 2010

2010-08-31T11:28:49Z

←Older revision		Revision as of 11:28, 31 August 2010
Line 8:		Line 8:
	*[[IGEM:Imperial/2010/Vectors_team \| Vectors team (Kyasha and Kirill)]]		*[[IGEM:Imperial/2010/Vectors_team \| Vectors team (Kyasha and Kirill)]]
	*[[IGEM:Imperial/2010/Surface_Protein_team \| Surface Protein team (Harriet and Florian)]]		*[[IGEM:Imperial/2010/Surface_Protein_team \| Surface Protein team (Harriet and Florian)]]
		+
		+	'''Key updates:'''
		+
		+	* ''XylE characterization'': During the next week (week 9) we will start characterization of XylE. Purification of XylE-His tagged from Jeremy's cultures will be a first step in direction of in-vitro assays. Upon assembly of the GFP-XylE fusion protein this week, protein purification will provide us with both XylE and GFP-XylE for in vitro testing. Important kinetic data will hopefully be obtained during the next 7 days.
		+

Wolfgang Pernice at 11:23, 31 August 2010

2010-08-31T11:23:57Z

←Older revision		Revision as of 11:23, 31 August 2010
Line 9:		Line 9:
	*[[IGEM:Imperial/2010/Surface_Protein_team \| Surface Protein team (Harriet and Florian)]]		*[[IGEM:Imperial/2010/Surface_Protein_team \| Surface Protein team (Harriet and Florian)]]

-	~~LINKS to shedule history (?)~~	+

	==Assembly strategy==		==Assembly strategy==

Wolfgang Pernice at 11:01, 24 August 2010

2010-08-24T11:01:28Z

←Older revision		Revision as of 11:01, 24 August 2010
Line 37:		Line 37:

	* ComE promoter system - recap on mode of function.		* ComE promoter system - recap on mode of function.
		+
		+	[[Image:Primers1.1.docx]]

Wolfgang Pernice at 15:29, 18 August 2010

2010-08-18T15:29:57Z

←Older revision		Revision as of 15:29, 18 August 2010
Line 5:		Line 5:
	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.

-	~~LINKS to lab teams.~~	+	*[[IGEM:Imperial/2010/XylE_team \| XylE team (Maddie and Nicolas)]]
		+	*[[IGEM:Imperial/2010/Vectors_team \| Vectors team (Kyasha and Kirill)]]
		+	*[[IGEM:Imperial/2010/Surface_Protein_team \| Surface Protein team (Harriet and Florian)]]

	LINKS to shedule history (?)		LINKS to shedule history (?)

Wolfgang Pernice at 16:36, 17 August 2010

2010-08-17T16:36:39Z

←Older revision		Revision as of 16:36, 17 August 2010
Line 1:		Line 1:
	=Strategy=		=Strategy=

-	~~==Assembly strategy and Lab-work==~~	+	Please refer to the assembly strategy for details.
-	Please refer to the ~~overall~~ 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.		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.
Line 9:		Line 8:

	LINKS to shedule history (?)		LINKS to shedule history (?)
		+
		+	==Assembly strategy==
		+
	[[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]]

Wolfgang Pernice at 16:35, 17 August 2010

2010-08-17T16:35:37Z

←Older revision		Revision as of 16:35, 17 August 2010
Line 9:		Line 9:

	LINKS to shedule history (?)		LINKS to shedule history (?)
		+	[[Image:Assembly strat 1.1 Pic.JPG\|Center\|1000px\|x600px\| Assembly strategy version 1.1]]

	==Wolfs checklist==		==Wolfs checklist==