IGEM:IMPERIAL/2007/Projects/Experimental Design

• Introduction
• Phase 1
• Phase 2
• Phase 3
• Notes

We have identified three phases of experiments for each of the project:

• Phase 1 - Intial testing of the promoters and test constructs in vitro
• Phase 2 - Testing/Validation of the test constructs
• Phase 3 - Testing/Validation of final constructs

In addition any overlap between the experiments of the projects has been identified and considered when making the experimental design.

Status

• Several Problems have been identified, we have begun to address them. Link here to problems.
• We are currently trying to improve our methodology to get better results. Link to improving our Methodology
• Problems with dsRED, it has failed quality control
• Currently trying to purify LuxR and GFP.

Time table

General timetable

	Week 8	Week 9	Week 10
Monday	-	Refine the wiki	Planning the week
Tuesday		Prepare GFP solution-calibration Construct Calibration Curve Refine the wiki	Testing to reduce evaporation
Wednesday	Test Home made cell extract Test varying levels of cell extract	PROBLEM-Wrong GFP!!!!	24hr experiment with pTet using paraffin oil
Thursday
Friday	Prepare GFP solution-calibration Construct Calibration Curve

CBD

	Week 8	Week 9	Week 10
Monday	-
Tuesday	Phase 1 - test pT7
Wednesday
Thursday	Test 20 and 37 °C, Test GFP degradation at 20 and 37 °C		24 Hour Exp Room Temp
Friday	Second day testing 20 and 37 °C		Define the thresholds of temperature

ID

	Week 8	Week 9	Week 10
Monday	-		Purify LuxR
Tuesday	test pLux in-vitro - 1nM AHL	Refine AHL concentrations range 25°C Purify LuxR if plasmid arrives	Purification of LuxR - problems
Wednesday		Purify LuxR if plasmid arrives	problems with LuxR pufification
Thursday		Clone in plasmid
Friday	Test construct with 5 AHL concentrations - 25 °C	Begin purification of LuxR	Test Purified LuxR with construct 2??