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.

Time table

Testing of constructs and cell extracts

	Week 8	Week 9	Week 10
Monday	-	Refine the wiki	Planning the week DsRED arrives
Tuesday		Prepare GFP solution-calibration Construct Calibration Curve Refine the wiki	Prepare DsRED constructs (Ask Ben)
Wednesday	Test Home made cell extract Test varying levels of cell extract	PROBLEM-Wrong GFP!!!!	Purify GFP Prepare DsRED constructs
Thursday			Purify GFP Prepare DsRED constructs
Friday	Prepare GFP solution-calibration Construct Calibration Curve		Purify GFP + Test Concentration Prepare DsRED constructs

CBD

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

ID

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

Future Tests

(CE:Number of Cell extracts needed)

1. Calibration curve GFP (CE:10)
2. Calibration curve dsRED (CE:10)
3. ID - Final Testing of AHL for dsRED (CE:20)
4. CBD - Steep Gradient dsRED- ( Ask Alex about final app)
   • Slow Gradient dsRED
   • Final Test Temp range for DsREd(CE:20)

Total Cell Extracts Reactions Required For Project

• Week 10: 20
• Future testing: 60
• In Stock: 72 Reactions

Dependencies

1. DsRed construct ( Tuesday)
2. Maxi Preps:
   • pLux Construct 2 - Wait for intial testing
   • Negative Control - Constuct rbs:GFP:Terminator, No promotoer