IGEM:IMPERIAL/2007/Projects/Cell by date/TestingValidation: Difference between revisions
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</calendar> | </calendar> | ||
== | =='''1. Initial Promoter Testing - IN VIVO''' == | ||
===1 | <br> | ||
'''Constructs:''' pTet-GFP, pT7-GFP, pcI-GFP | ===1.1 Initial testing for Cell by Date=== | ||
Test to see if construct will express ''in | {| 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 ] | |||
Test to see if construct will express ''in vivo''. Experiments carried out at 30°C in an incubator. | |||
<br> | |||
'''Aims:''' | '''Aims:''' | ||
*To determine if construct expresses ''in | *To determine if construct expresses ''in vivo'' | ||
<br> | <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 | *50μl in vitro chassis | ||
*DNA concentration | *DNA concentration | ||
*Temperature stable at 30°C | |||
'''Variables | </div> | ||
< | |||
<span class="_toggler_toggle-item1-1-2">'''[+] Variables'''</span> | |||
<div class="toggle1-1 item1-1-2" style="display:none;"> | |||
*Rate of GFP synthesis | *Rate of GFP synthesis | ||
*Life span of chassis | *Life span of chassis | ||
*Response time | *Response time | ||
</div> | |||
'''Sampling | |||
< | <span class="_toggler_toggle-item1-1-3">'''[+] Sampling'''</span> | ||
<div class="toggle1-1 item1-1-3" style="display:none;"> | |||
*Every | *Every 5 minutes. | ||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
</div> | |||
'''Controls | |||
< | <span class="_toggler_toggle-item1-1-4">'''[+] Controls'''</span> | ||
<div class="toggle1-1 item1-1-4" style="display:none;"> | |||
*Negative Control: | *Negative Control: E.Coli cultures without GFP expressing machinery | ||
*Positive Control: | *Positive Control: Diluted GFP in well | ||
</div> | |||
<br> | |||
<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> | |||
==Phase 2: Characterizing Specific Construct== | ==Phase 2: Characterizing Specific Construct== |
Revision as of 01:44, 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>
1. Initial Promoter Testing - IN VIVO
1.1 Initial testing for Cell by Date
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
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