IGEM:IMPERIAL/2007/Projects/Experimental Design: Difference between revisions
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Maira Tariq (talk | contribs) (→ID) |
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====Proposed plan of action==== | ====Proposed plan of action==== | ||
#'''DsRed express''' - prioritise cloning and testing the constructs with DsRed Express | #'''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 | #'''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 | #*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 | |||
Revision as of 01:24, 7 September 2007
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 | |
| Tuesday | Prepare GFP solution-calibration Construct Calibration Curve Refine the wiki |
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| Wednesday | Test Home made cell extract Test varying levels of cell extract |
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| Thursday | |||
| Friday | Prepare GFP solution-calibration Construct Calibration Curve |
CBD
| Week 8 | Week 9 | Week 10 | |
| Monday | - | Exp with 15 and 25 °C | |
| Tuesday | Phase 1 - test pT7 | Exp with 30 and 40 °C, continue measuring 15 and 25 °C | |
| Wednesday | Exp with 10 and 45 °C, continue measuring 30 and 40 °C | ||
| Thursday | Test 20 and 37 °C, Test GFP degradation at 20 and 37 °C |
Test suitable step increases | |
| Friday | Second day testing 20 and 37 °C | Test suitable gradient of temperature, minimum working temp - maximum working temp |
ID
| Week 8 | Week 9 | Week 10 | |
| Monday | - | ||
| Tuesday | test pLux in-vitro - 1nM AHL | Refine AHL concentrations range 25°C Purify LuxR if plasmid arrives |
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| Wednesday | Choose a range of AHL concentrations range 37°C Purify LuxR if plasmid arrives |
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| Thursday | Test purified LuxR | ||
| Friday | Test construct with 5 AHL concentrations - 25 °C | Test purified 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
