IGEM:IMPERIAL/2007/Projects/Cell by date/TestingValidation

Cell by Date: Testing/Validation

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Lab Notebook

• Week 5 (6 Aug): Building Constructs
  
• Week 6 (13 Aug): Building Constructs / Phase 1
  
• Week 7 (20 Aug): Phase 1 / Phase 2
  
• 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

1.1 Initial testing for Cell by Date in vivo

Overall Status
Results uploaded !
Construct	Status
pTet-GFP	Tested - 16/08/2007
pT7-GFP	Tested - 17/08/2007
pCI-GFP	No Longer Testing - Problem with Biobricks

Constructs: pTet-GFP , pT7-GFP ,pcI-GFP

Test to see if construct will express in vivo. Experiments carried out at 30°C in an incubator.

Aims:

• To determine if construct expresses in vivo

	Protocol
Results

[+] Constant Conditions

• 50μl in vitro chassis
• DNA concentration
• Temperature stable at 30°C

[+] Variables

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

[+] Sampling

• Every 5 minutes.

Repetition:

• 3 repeats

[+] Controls

• Negative Control: E.Coli cultures without GFP expressing machinery
• Positive Control: Diluted GFP in well

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1.2 Initial testing for Cell by Date in vitro

Overall Status
Construct	Status
pTet-GFP	Initial Testing - 21/08/2007
pT7-GFP	Initial Testing - 21/08/2007
pCI-GFP	No Longer Testing - Problem with Biobricks

Constructs: pTet-GFP, pT7-GFP , pcI-GFP


Aims:
Experiment 1 :

• Determine the DNA concentration and purity. From this determine volume of the DNA needed to add to the in vitro expression systems.

	Protocol
Results

Experiment 2:

• To determine which constructs for cell by date expresses in vitro
• To test the pTet and pcI constructs in commercial S30 E.coli cell extract and home made S30 extract
• To test the pT7 construct in commercial S30 T7 cell extract and home made S30 extract.
• To investigate optimum volume of home made S30 extract
• To investigate the differences between home made and commercially bought S30 extract. This is in terms of rates of expression, length of expression and total output.

Status:

• Home made extract on hold until next week, this is because of problem that there was no amino acid or tRNA supplements added. We are carrying on with the commercial extracts for our testing
• DNA Constructs: Purified samples of pTet+pT7 available Friday, pcI available next week.
• T7 Commercial extract arrived
• E.coli commercial extract to arrive Friday

[+] Constant Conditions

• 2ug DNA added
• 37°C

[+] Variables

• Type of cell extract used : Commercial or home made
• For home made extract we will vary the volumes used

[+] Sampling

• 30minutes

Repetition:

• 2 repeats

[+] Controls

• Negative Control: In vitro system only to measure background fluorescence
• Positive Control: In vitro system with purified GFP added

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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].
Aims:

• To determine [GFP] vs Fluorescence

Conditions:
[show][hide]

• 50μl in vitro chassis
• DNA added
  • Do we need DNA, as it may absorb some of the fluorescence
• 25^oC

Variables:
[show][hide]

• [GFP] added
• Fluorescence

Sampling:
[show][hide]

• Measure after addition of GFP to minimize degradation of GFP

Repetition:

• 3 repeats

Controls:
[show][hide]

• 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:
[show][hide]

• 50μl in vitro chassis
• [GFP] added

Variables:
[show][hide]

• Temperature range: 4^oC, 15^oC, 25^oC, 30^oC, 37^oC, 50^oC
• Range may change based upon the initial testing; will only test ranges that in vitro is stable at.
• Degradation of GFP

Sampling:
[show][hide]

• Every 15 minutes.

Repetition:

• 3 repeats

Controls:
[show][hide]

• 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: 4^oC, 15^oC, 25^oC, 30^oC, 37^oC, 50^oC
• To determine specific life span at each temperature range.
• To determine the maximum rate of GFP produced at each temperature range.

Conditions:
[show][hide]

• 50μl in vitro chassis
• DNA concentration

Variables:
[show][hide]

• Rate of GFP synthesis
• Life span of chassis
• Response time
• Temperature: 4^oC, 15^oC, 25^oC, 30^oC, 37^oC, 50^oC

Sampling:
[show][hide]

• Every 15 minutes.

Repetition:

• 3 repeats

Controls:
[show][hide]

• 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:
[show][hide]

• 50μl in vitro chassis
• DNA concentration

Variables:
[show][hide]

• Rate of GFP synthesis
• Life span of chassis
• Response time
• Temperature change 4^oC, 25^oC, 37^oC
  • 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:
[show][hide]

• Every 30 min interval.
• Every 15 minutes for 2 hours after change in temperature.
• Every 30 minutes thereafter.

Repetition:

• 3 repeats

Controls:
[show][hide]

• 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:
[show][hide]

• 50μl in vitro chassis
• DNA concentration

Variables:
[show][hide]

• Rate of GFP synthesis
• Life span of chassis
• Response time
• Temperature change 4^oC, 25^oC, 37^oC
  • Type of gradients: Steep (5 min)
  • Temperature change : from 4°C to 25^oC, 37^oC and vice versa
  • Time period before increment: 30 min, 1h, 2h,

Sampling:
[show][hide]

• Every 30 min interval.
• Every 15 minutes for 2 hours after change in temperature.
• Every 30 minutes thereafter.

Repetition:

• 3 repeats

Controls:
[show][hide]

• 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:
[show][hide]

• 50μl in vitro chassis
• DNA concentration

Variables:
[show][hide]

• Rate of GFP synthesis
• Life span of chassis
• Response time
• Temperature change 4^oC, 25^oC, 37^oC
  • Type of gradients: Pulse(5 min, 1 hour)
  • Temperature change : from 4°C to 25^oC, 37^oC 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:
[show][hide]

• Every 30 min interval.
• Every 15 minutes for 2 hours after change in temperature.
• Every 30 minutes thereafter.

Repetition:

• 3 repeats

Controls:
[show][hide]

• Negative Control: In vitro system only
• Positive Control: In vitro system with purified GFP added