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

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*'''Week 9 (3 Sep):''' <br>
*'''Week 9 (3 Sep):''' <br>
*'''Week 10 (10 Sep):''' <br>
*'''Week 10 (10 Sep):''' <br>


<calendar>
<calendar>
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<br>
<br>
{{hide|
{{hide|
*Independent variables :
*Rate of GFP synthesis
*Dependent variables:
*Life span of chassis
**Rate of GFP synthesis
*Response time
**Life span of chassis
**Response time
}}
}}
'''Sampling:'''
'''Sampling:'''
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<br>
<br>
{{hide|
{{hide|
*Negative Control: In vitro system with no AHL added
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
*Positive Control: In vitro system with purified GFP added
}}
}}
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'''Aims:'''
'''Aims:'''
*To determine [GFP] vs Fluorescence  
*To determine [GFP] vs Fluorescence  
'''Constant Conditions:'''<br>
'''Conditions:'''<br>
{{hide|
{{hide|
*50μl in vitro chassis
*50μl in vitro chassis
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<br>
<br>
{{hide|
{{hide|
*Independent variables :
*[GFP] added
**[GFP] added
*Fluorescence
*Dependent variables:
**Fluorescence
}}
}}
'''Sampling:'''
'''Sampling:'''
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<br>
<br>
{{hide|
{{hide|
*Negative Control:  
*Negative Control: In vitro system only
**No GFP is added to an in vitro chassis
**No GFP is added to an in vitro chassis
}}
}}
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{{hide|
{{hide|
*50μl in vitro chassis
*50μl in vitro chassis
*GFP added....
*[GFP] added
}}
}}
'''Variables:'''
'''Variables:'''
<br>
<br>
{{hide|
{{hide|
*Independent variables :
*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
**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>
*Range may change based upon the initial testing; will only test ranges that in vitro is stable at.  
**Range may change based upon the initial testing; will only test ranges that in vitro is stable at.  
*Degradation of GFP
*Dependent variables:
**Degradation of GFP
}}
}}
'''Sampling:'''
'''Sampling:'''
<br>
<br>
{{hide|
{{hide|
*Every 5 minutes.
*Every 15 minutes.
Repetition:
Repetition:
*3 repeats
*3 repeats
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<br>
<br>
{{hide|
{{hide|
*Negative Control: No GFP is added to an in vitro chassis
*Negative Control: In vitro system only
*Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature
*Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature
(CHECK)
}}
}}
<br>
<br>
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Test to determine the operating range of the preferred construct ''in vitro''. Experiments carried out across various temperatures.<br>
Test to determine the operating range of the preferred construct ''in vitro''. Experiments carried out across various temperatures.<br>
'''Aims:'''
'''Aims:'''
*To determine if construct expresses ''in vitro'' at temperatures of: 4°C, 15°C, 25°C, 37°C, 45°C, 60°C.
*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
'''Conditions:'''
'''Conditions:'''
<br>
<br>
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<br>
<br>
{{hide|
{{hide|
*Independent variables :
*Rate of GFP synthesis
*Dependent variables:
*Life span of chassis
**Rate of GFP synthesis
*Response time
**Life span of chassis
*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
**Response time
**Temperature: 4°C, 15°C, 25°C, 37°C, 45°C, 60°C
}}
}}
'''Sampling:'''
'''Sampling:'''
<br>
<br>
{{hide|
{{hide|
*Every 5 minutes.
*Every 15 minutes.
Repetition:
Repetition:
*3 repeats
*3 repeats
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<br>
<br>
{{hide|
{{hide|
*Negative Control: In vitro system with no AHL added
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
*Positive Control: In vitro system with purified GFP added
}}
}}
<br>
<br>

Revision as of 05:31, 13 August 2007

Cell by Date: Implementation



Lab Notebook

  • Week 5 (6 Aug): Phase 1
  • Week 6 (13 Aug): 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 under room temperature 25°C.
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:

  • 25 oC
  • 50μl in vitro chassis
  • DNA concentration

Variables:

  • Rate of GFP synthesis
  • Life span of chassis
  • Response time

Sampling:

  • Every 5 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

Constant 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

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


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