IGEM:IMPERIAL/2007/Projects/Cell by date/TestingValidation: Difference between revisions

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<br>
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==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].<br>
'''Aims:'''
*To determine [GFP] vs Fluorescence
'''Conditions:'''<br>
{{hide|
*50μl in vitro chassis
*DNA added
**Do we need DNA, as it may absorb some of the fluorescence
*25<sup>o</sup>C
}}
'''Variables:'''
<br>
{{hide|
*[GFP] added
*Fluorescence
}}
'''Sampling:'''
<br>
{{hide|
*Measure after addition of GFP to minimize degradation of GFP
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
**No GFP is added to an in vitro chassis
}}
<br>


===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.<br>
'''Aims:'''
*To determine the half life of GFP for a range of temperatures
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*[GFP] added
}}
'''Variables:'''
<br>
{{hide|
*Temperature range: 4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>C
*Range may change based upon the initial testing; will only test ranges that in vitro is stable at.
*Degradation of GFP
}}
'''Sampling:'''
<br>
{{hide|
*Every 15 minutes.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature
}}
<br>
===3) Operating Temperature Range===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
Test to determine the operating range of the preferred construct ''in vitro''. Experiments carried out across various temperatures.<br>
'''Aims:'''
*To determine if construct expresses ''in vitro'' at temperatures of: 4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>C
*To determine specific life span at each temperature range.
*To determine the maximum rate of GFP produced at each temperature range.
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature: 4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>C
}}
'''Sampling:'''
<br>
{{hide|
*Every 15 minutes.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
===4) Varying Temperature Changes: Gentle Gradient===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
'''Aims:'''
*To determine the effects of fluorescence with reference to a gentle change in temperature from 4&deg;C to 37&deg;C and vice versa over different time periods.
*Provide results for modelling.
**Investigate k constant as a function of temperature and time.
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature change 4<sup>o</sup>C, 25<sup>o</sup>C, 37<sup>o</sup>C
**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:'''
<br>
{{hide|
*Every 30 min interval.
*Every 15 minutes for 2 hours after change in temperature.
*Every 30 minutes thereafter.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
===5) Varying Temperature Changes: Steep Gradient===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
'''Aims:'''
*To determine the effects of fluorescence with reference to a steep change in temperature from 4&deg;C to 37&deg;C and vice versa over different time periods.
*Provide results for modelling.
**Investigate k constant as a function of temperature and time.
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature change 4<sup>o</sup>C, 25<sup>o</sup>C, 37<sup>o</sup>C
**Type of gradients: Steep (5 min)
**Temperature change : from 4°C to 25<sup>o</sup>C, 37<sup>o</sup>C and vice versa
**Time period before increment: 30 min, 1h, 2h,
}}
'''Sampling:'''
<br>
{{hide|
*Every 30 min interval.
*Every 15 minutes for 2 hours after change in temperature.
*Every 30 minutes thereafter.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>


==Phase 3: Testing/Validation of Modelling Analysis==
==Phase 3: Testing/Validation of Modelling Analysis==

Revision as of 07:40, 14 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


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