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.
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 |
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 |
Meeting to sort the GFP crisis!
Options
- GFP
- AcGFP -
- GFP mut3b - need to get the purified form
- Ask around!
- RFP
- DsRed - wild type
- DsRed2 - can be visualied but has a long response time ~ 24hrs
- DsRed express - can be visualised with UV, has response time of ~ 7-8hrs
Proposed plan of action
- DsRed express - prioritise cloning and testing the constructs with DsRed Express
- Currently testing DsRed Express in-vitro (Calibration curve)
- GFP mut 3b - carry on looking for purified form so calibration and degradation curves can be derived
- Do research to find degradation and calibration data in literature
- DesRed 2 will be cloned just in case the other options don't work out
Flurometer
- Might get access to 24hr fluorometer - programmed data collection (biochem lab, level 6)
- Can carry out only the experiments at room temperature and degradation experiments