IGEM:IMPERIAL/2007/Projects/Cell by date/TestingValidation: Difference between revisions
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===1) Initial Testing=== | ===1) Initial Testing=== | ||
'''Constructs:''' pTet-GFP, pT7-GFP, pcI-GFP<br> | '''Constructs:''' pTet-GFP, pT7-GFP, pcI-GFP<br> | ||
Test to see if construct will express ''in vitro''. Experiments carried out at | Test to see if construct will express ''in vitro''. Experiments carried out at 4°C in the cold room, 25°C and 37°C in an incubator.<br> | ||
'''Aims:''' | '''Aims:''' | ||
*To determine if construct expresses ''in vitro'' | *To determine if construct expresses ''in vitro'' | ||
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<br> | <br> | ||
{{hide| | {{hide| | ||
*50μl in vitro chassis | *50μl in vitro chassis | ||
*DNA concentration | *DNA concentration | ||
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<br> | <br> | ||
{{hide| | {{hide| | ||
*Temperature change 4°C, 25°C, 37°C | |||
*Rate of GFP synthesis | *Rate of GFP synthesis | ||
*Life span of chassis | *Life span of chassis | ||
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<br> | <br> | ||
==Phase 2: Characterizing Specific Construct== | ==Phase 2: Characterizing Specific Construct== |
Revision as of 16:33, 13 August 2007
Cell by Date: Testing/Validation
Lab Notebook
- Week 5 (6 Aug): Building Constructs
- Week 6 (13 Aug): Building Constructs / Phase 1
- Week 7 (20 Aug):
- Week 8 (27 Aug):
- Week 9 (3 Sep):
- Week 10 (10 Sep):
<calendar>
name=iGEM:IMPERIAL/2007/Notebook
date=2007/09/15
view=threemonths
format=%name/%year-%month-%day
weekstart=7
</calendar>
Phase 1: Initial Testing
1) Initial Testing
Constructs: pTet-GFP, pT7-GFP, pcI-GFP
Test to see if construct will express in vitro. Experiments carried out at 4°C in the cold room, 25°C and 37°C in an incubator.
Aims:
- To determine if construct expresses in vitro
- To get approximations of: life span, response time and rate of GFP synthesized.
- To determine whether the constructs or the in vitro need to be optimised.
Conditions:
- 50μl in vitro chassis
- DNA concentration
Variables:
- Temperature change 4°C, 25°C, 37°C
- Rate of GFP synthesis
- Life span of chassis
- Response time
Sampling:
- Every 15 minutes.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
Phase 2: Characterizing Specific Construct
1) Calibration Curve for GFP
Test to determine the relationship between fluorescence and in vitro concentration of GFP. To test this purified samples of known [GFP] are added to in vitro chassis and the fluorescence measured. From this a calibration curve of [GFP] vs Fluorescence can be made. This can be used for data analysis to convert fluorescence into a [GFP].
Aims:
- To determine [GFP] vs Fluorescence
Conditions:
- 50μl in vitro chassis
- DNA added
- Do we need DNA, as it may absorb some of the fluorescence
- 25oC
Variables:
- [GFP] added
- Fluorescence
Sampling:
- Measure after addition of GFP to minimize degradation of GFP
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- No GFP is added to an in vitro chassis
2) Degradation Time of GFP
Test the half life of GFP protein in an in vitro chassis. To test this a purified sample of known [GFP] are added to an in vitro chassis, then fluorescence will be measured at regular time intervals. The fluorescence will be converted into GFP molecules using the calibration curve. This will give; degradation of GFP as a function of time, from this the half life of GFP can be obtained. In addition, temperature may affect the half life of GFP and so the half life will be measured for an appropriate temperature range.
Aims:
- To determine the half life of GFP for a range of temperatures
Conditions:
- 50μl in vitro chassis
- [GFP] added
Variables:
- Temperature range: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
- Range may change based upon the initial testing; will only test ranges that in vitro is stable at.
- Degradation of GFP
Sampling:
- Every 15 minutes.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature
3) Operating Temperature Range
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Test to determine the operating range of the preferred construct in vitro. Experiments carried out across various temperatures.
Aims:
- To determine if construct expresses in vitro at temperatures of: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
- To determine specific life span at each temperature range.
- To determine the maximum rate of GFP produced at each temperature range.
Conditions:
- 50μl in vitro chassis
- DNA concentration
Variables:
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
Sampling:
- Every 15 minutes.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
4) Varying Temperature Changes: Gentle Gradient
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Aims:
- To determine the effects of fluorescence with reference to a gentle change in temperature from 4°C to 37°C and vice versa over different time periods.
- Provide results for modelling.
- Investigate k constant as a function of temperature and time.
Conditions:
- 50μl in vitro chassis
- DNA concentration
Variables:
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature change 4oC, 25oC, 37oC
- Type of gradients: Gentle(1 hour)
- Temperature change : from 4°C to 25°C, 37°C and vice versa
- Time period before increment: 30 min, 1h, 2h (to be done in parallel)
Sampling:
- Every 30 min interval.
- Every 15 minutes for 2 hours after change in temperature.
- Every 30 minutes thereafter.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
5) Varying Temperature Changes: Steep Gradient
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Aims:
- To determine the effects of fluorescence with reference to a steep change in temperature from 4°C to 37°C and vice versa over different time periods.
- Provide results for modelling.
- Investigate k constant as a function of temperature and time.
Conditions:
- 50μl in vitro chassis
- DNA concentration
Variables:
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature change 4oC, 25oC, 37oC
- Type of gradients: Steep (5 min)
- Temperature change : from 4°C to 25oC, 37oC and vice versa
- Time period before increment: 30 min, 1h, 2h,
Sampling:
- Every 30 min interval.
- Every 15 minutes for 2 hours after change in temperature.
- Every 30 minutes thereafter.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
Phase 3: Testing/Validation of Modelling Analysis
1) Testing/Validation of Device
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Aims:
- To validate the predictions of modelling analysis.
- To fine-tune the in vitro system
Conditions:
- 50μl in vitro chassis
- DNA concentration
Variables:
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature change 4oC, 25oC, 37oC
- Type of gradients: Pulse(5 min, 1 hour)
- Temperature change : from 4°C to 25oC, 37oC and vice versa
- Time period before increment: 30 min, 1h, 2h (subject to change)
- Temperature period maintained: 5 min, 15 min, 30 min, 1h, 2h
Sampling:
- Every 30 min interval.
- Every 15 minutes for 2 hours after change in temperature.
- Every 30 minutes thereafter.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added