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

Time table

Testing of constructs and cell extracts

	Week 8	Week 9	Week 10
Monday	-	Refine the wiki	Planning the week
Tuesday		Prepare GFP solution-calibration Construct Calibration Curve Refine the wiki	DsRED arrives
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 Investigate Evap Problem
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
Wednesday		Purify LuxR if plasmid arrives	Test Purified LuxR with construct 2
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