IGEM:IMPERIAL/2007/Projects/Experimental Design
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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 beguun to address them. Link here to problems.
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 |
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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 |
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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)
- Calibration curve GFP (CE:10)
- Calibration curve dsRED (CE:10)
- ID - Final Testing of AHL for dsRED (CE:20)
- 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
- DsRed construct ( Tuesday)
- Maxi Preps:
- pLux Construct 2 - Wait for intial testing
- Negative Control - Constuct rbs:GFP:Terminator, No promotoer