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

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===Experimental design===
==Lab Notebook==


*Temperature increases the rate of reactions - increased temperature means faster gene expression over a given time.
*'''Week 5 (6 Aug):''' Building Constructs<br>
*System only deals with protein synthesis and its reporter function - a visual signal.
*'''Week 6 (13 Aug):''' Building Constructs / Phase 1<br>
*Coupling firefly genes with that of a well-characterized consitutive promoter, response is good.
*'''Week 7 (20 Aug):''' Phase 1 / Phase 2<br>
*We will then calibrate the synthesis of fluorescent protein (which is easier as opposed to calibrating cell density) with that of the predicted shelf-life (date label).
*'''Week 8 (27 Aug):''' <br>
**Assuming the cold chain process only occurs at 4oC, it will lead to the predicted outcome.
*'''Week 9 (3 Sep):''' <br>
**Any temperature increases, and over a threshold would lead to over the range fluorescence.
*'''Week 10 (10 Sep):''' <br>
**Degradation of protein is minimal with fine-tuning of cell free medium characterization
**Proof of principle of the application of CFE.




===Fluorescent Signal===
<calendar>
name=iGEM:IMPERIAL/2007/Notebook
date=2007/09/15
view=threemonths
format=%name/%year-%month-%day
weekstart=7
</calendar>


* The first problem is that a fluorescent signal will only be visible if a cell population is at a certain density. This is because there needs to be a great enough density of cells expressing a fluorescent protein so that there is a strong enough signal to be visible by eye. This will be a problem if the cells are exposed to varying conditions early on in the sell by date when cell population is low.
=='''Phase 1: Initial Testing''' ==
* The fluorescent proteins expressed will also need to be relatively stable from degradation. This is because if we initiate the expression of the fluorescent proteins then we want the signal to be sustained so that our 'cell-by-date' has a memory.
<br>
**A further point on this is that we can't use GFP as a reporter in hypoxic conditions GFPHypoxic
===1.1 Initial testing for Cell by Date in vivo===
**Possible solution to this problem is to use a reporter with a long Half-Life so that there is enough time for someone to see the signal being outputted by our system. EGFP Seems to be one such reporter having a half life that may exceed 24hours1. Enocding vector found for EGFP that places it under control of Plac promoter4. DsRed seems to be another possible reporter to use3.
{| width= 30% border="2" style="background-color:#ABCDEF;" align="right"
| colspan="2" align="center"  style="font-style:Bold; font-size:140%;" | Overall Status
|-
| colspan="2" align="center" style="color:#9933FF ; font-style:Bold; font-size:140%;" | Results uploaded !
|-
|align="center"  style="font-size:100%;" | '''Construct'''
|align="center"  style="font-size:100%;" | '''Status'''
|-
|style="font-style:Bold;" align="center" | pTet-GFP
|style="color:Green ; font-style:Bold; font-size:110%;" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | Tested - 16/08/2007 ]]
|-
|style="font-style:Bold;" align="center"| pT7-GFP
|style="color:Blue ; font-style:Bold; font-size:110%;" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-17 | Tested - 17/08/2007 ]]
|-
|style="font-style:Bold;" align="center"| pCI-GFP
|style="color:Red ; font-style:Bold; font-size:110%;" align="center" | No Longer Testing - Problem with Biobricks
|}
<br>
'''Constructs:''' [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP ], [http://parts.mit.edu/registry/index.php/Part:BBa_E7104 pT7-GFP ] ,[http://parts.mit.edu/registry/index.php/Part:BBa_I719022 pcI-GFP ]


*In order to avoid complication in design we have opted to just use one reporter, RFP, instead of using two eg. transition from GFP to RFP when problems occour. This is due to time constraints - it make take time for the RFP to overcome the GFP.
 
Test to see if construct will express ''in vivo''. Experiments carried out at 30&deg;C in an incubator.
 
<br>
'''Aims:'''
*To determine if construct expresses ''in vivo''
<br>
{| border="0" align="right" style="font-style:Bold; font-size:140%; color:red;"
| align="left" width="170px" |
| align="right"  style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.1 |'''Protocol''']]
|-
| colspan=2 align="right"  style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 1.1 |'''Results''']]
|}
 
 
<span class="_toggler_toggle-item1-1-1">'''[+] Constant Conditions'''</span>
<div class="toggle1-1 item1-1-1" style="display:none;">
*50μl in vitro chassis
*DNA concentration
*Temperature stable at 30&deg;C
</div>
 
<span class="_toggler_toggle-item1-1-2">'''[+] Variables'''</span>
<div class="toggle1-1 item1-1-2" style="display:none;">
*Rate of GFP synthesis
*Life span of chassis
*Response time
</div>
 
<span class="_toggler_toggle-item1-1-3">'''[+] Sampling'''</span>
<div class="toggle1-1 item1-1-3" style="display:none;">
*Every 5 minutes.
Repetition:
*3 repeats
</div>
 
<span class="_toggler_toggle-item1-1-4">'''[+] Controls'''</span>
<div class="toggle1-1 item1-1-4" style="display:none;">
*Negative Control: E.Coli cultures without GFP expressing machinery
*Positive Control: Diluted GFP in well
</div>
 
<div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle1-1 _toggler-toggler1-1a _toggler-toggler1-1b toggler1-1a">Show All Details</span><span class="_toggler_hide-toggle1-1 _toggler-toggler1-1a _toggler-toggler1-1b toggler1-1b" style="display:none;">Hide All Details</span></div>
 
<br><br><br>
<HR SIZE=2 WIDTH=90%><br>
 
<br>
===1.2 Initial testing for Cell by Date in vitro===
{| width= 30% border="2" style="background-color:#ABCDEF;" align="right"
| colspan="2" align="center"  style="font-style:Bold; font-size:140%;" | Overall Status
|-
|align="center"  style="font-size:100%;" | '''Construct'''
|align="center"  style="font-size:100%;" | '''Status'''
|-
|style="font-style:Bold;" align="center" | pTet-GFP
|style="color:Green ; font-style:Bold; font-size:110%;" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-21 | Initial Testing - 21/08/2007 ]]
|-
|style="font-style:Bold;" align="center"| pT7-GFP
|style="color:Blue ; font-style:Bold; font-size:110%;" align="center" |  [[IGEM:IMPERIAL/2007/Notebook/2007-8-21 | Initial Testing - 21/08/2007 ]]
|-
|style="font-style:Bold;" align="center"| pCI-GFP
|style="color:Red ; font-style:Bold; font-size:110%;" align="center" | No Longer Testing - Problem with Biobricks
|}
 
<br>
<br>
<br>
'''Constructs:''' [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP],  [http://parts.mit.edu/registry/index.php/Part:BBa_E7104 pT7-GFP] , [http://parts.mit.edu/registry/index.php/Part:BBa_I719022 pcI-GFP]
<br>
<br>
<br>
'''Aims:'''<br>
''Experiment 1 :''
*Determine the DNA concentration and purity. From this determine volume of the DNA needed to add to the ''in vitro'' expression systems.
{| border="0" align="right" style="font-style:Bold; font-size:140%; color:red;"
| align="left" width="170px" |
| align="right"  style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 2.1 |'''Protocol''']]
|-
| colspan=2 align="right"  style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.1 |'''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:'''
*<font color=red> 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 </font>
*DNA Constructs: Purified samples of pTet+pT7 available Friday, pcI available next week.
*T7 Commercial extract arrived
*E.coli commercial extract to arrive Friday
 
 
<span class="_toggler_toggle-item2-1-1">'''[+] Constant Conditions'''</span>
<div class="toggle2-1 item2-1-1" style="display:none;">
*2ug DNA added
*37&deg;C
</div>
 
<span class="_toggler_toggle-item2-1-2">'''[+] Variables'''</span>
<div class="toggle2-1 item2-1-2" style="display:none;">
*Type of cell extract used : Commercial or home made
*For home made extract we will vary the volumes used
</div>
 
<span class="_toggler_toggle-item2-1-3">'''[+] Sampling'''</span>
<div class="toggle2-1 item2-1-3" style="display:none;">
*30minutes
Repetition:
*2 repeats
</div>
 
<span class="_toggler_toggle-item2-1-4">'''[+] Controls'''</span>
<div class="toggle2-1 item2-1-4" style="display:none;">
*Negative Control: In vitro system only to measure background fluorescence
*Positive Control: In vitro system with purified GFP added
</div>
 
<div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle2-1 _toggler-toggler2-1a _toggler-toggler2-1b toggler2-1a">Show All Details</span><span class="_toggler_hide-toggle2-1 _toggler-toggler2-1a _toggler-toggler2-1b toggler2-1b" style="display:none;">Hide All Details</span></div>
 
<br><br><br>
<HR SIZE=2 WIDTH=90%><br>
 
==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==
===1) Testing/Validation of Device===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
'''Aims:'''
*To validate the predictions of modelling analysis.
*To fine-tune the ''in vitro'' system
'''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: Pulse(5 min, 1 hour)
**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 (subject to change)
**Temperature period maintained: 5 min, 15 min, 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>

Latest revision as of 01:50, 28 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): 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

[+] Variables

[+] Sampling

[+] Controls

Show All Details






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

[+] Variables

[+] Sampling

[+] Controls

Show All Details





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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