IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1: Difference between revisions
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=='''3.1 Investigate optimum counting time for fluorodetector'''== | |||
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A test to see what is the optimum counting time to have during our experiments. The counting time is the time the fluorometer detector stays on top of each well. A small window time will only account for very discrete levels of fluorescence. These might include sudden spikes of radiation since fluorescence is not a uniform process. Hence we will get variation between samples of equal expression rates. A larger counting time results in a larger window size and hence a more average reading is taken from each sample smoothing out the variation due to the randomness of fluorescence emition. | A test to see what is the optimum counting time to have during our experiments. The counting time is the time the fluorometer detector stays on top of each well. A small window time will only account for very discrete levels of fluorescence. These might include sudden spikes of radiation since fluorescence is not a uniform process. Hence we will get variation between samples of equal expression rates. A larger counting time results in a larger window size and hence a more average reading is taken from each sample smoothing out the variation due to the randomness of fluorescence emition. <br> | ||
Care must be taken however because larger counting times will lead to faster fluorescence bleaching. A comprimise between the two must be found. | |||
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| align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol | | align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 3.1 |'''Protocol''']] | ||
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| colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results | | colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 3.1 |'''Results''']] | ||
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'''Aims:'''<br> | '''Aims:'''<br> | ||
Determine the optimum counting time for the fluorometer. | |||
Avoid fluorescence bleaching. | |||
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<br> | <br> | ||
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<span class="_toggler_toggle- | <span class="_toggler_toggle-item3-1-1">'''[+] Constant Conditions'''</span> | ||
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* | *Temperature: 25 <sup>o</sup>C (Room temperature) | ||
* | *200 µl aliquots from pTet culture in each well | ||
</div> | </div> | ||
<span class="_toggler_toggle- | <span class="_toggler_toggle-item3-1-2">'''[+] Variables'''</span> | ||
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* | *Vary the counting time of the fluorometer from 0.15 sec to 0.9 sec. | ||
* | *The counting times tested will be : 0.15, 0.30, 0.45, 0.60, 0.75, 0.90 sec | ||
</div> | </div> | ||
<span class="_toggler_toggle- | <span class="_toggler_toggle-item3-1-3">'''[+] Sampling'''</span> | ||
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* | *Take readings every 5 minutes for 1 hour. | ||
Repetition: | Repetition: | ||
* | *4 repeats | ||
</div> | </div> | ||
<span class="_toggler_toggle- | <span class="_toggler_toggle-item3-1-4">'''[+] Controls'''</span> | ||
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*Negative Control: | *Negative Control: E.Coli cultures without GFP expressing machinery | ||
*Positive Control: | *Positive Control: Diluted GFP in well | ||
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Revision as of 17:56, 20 August 2007
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 | Waiting for construct |
Constructs: pTet-GFP (BBa_I13522), pT7-GFP (BBa_E7104) , pcI-GFP (BBa_I719022)
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
1.2 Initial testing for Infector Detector
Status | |
Planned for 22/8/07 | INCOMPLETE |
Construct: pTet - LuxR - pLux - GFP (BBa_T9002)
Test to see if construct will express in vivo. Experiments carried out in incubator at 30oC at a range of inducer concentrations. To do this, we induce E. coli cells transfected with the construct with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution.
Protocol | |
Results |
Aims:
- To determine if construct expresses GFP in vivo
[+] Constant Conditions
[+] Variables
[+] Sampling
[+] Controls
2. Initial Promoter Testing - IN VITRO
2.1 Initial Testing for Cell by Date
Overall Status | |
Construct | Status |
pTet-GFP | Initial Testing - 21/08/2007 |
pT7-GFP | Initial Testing - 21/08/2007 |
pCI-GFP | Waiting for construct |
Constructs: pTet-GFP (BBa_I13522), pT7-GFP (BBa_E7104) , pcI-GFP (BBa_I719022)
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
2.2 Initial testing for Infector Detector
Status | |
Initial Testing - 21/08/2007 | INCOMPLETE |
Construct: pTet - LuxR - pLux - GFP (BBa_T9002)
Test to see if construct will express in vitro. Experiments carried out at incubator at 37oC and under an inducer concentration that was shown to give a high induction in vivo.
Protocol | |
Results |
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.
Experiment 2:
- To determine if the infector detecter construct expresses in vitro
- To Test the construct in home made and in commercial e.coli cell extract
Status:
[+] Constant Conditions
[+] Variables
[+] Sampling
[+] Controls
3.1 Investigate optimum counting time for fluorodetector
Status | |
Planned for 21/08/2007 | INCOMPLETE |
Construct: pTet-GFP (BBa_I13522)
A test to see what is the optimum counting time to have during our experiments. The counting time is the time the fluorometer detector stays on top of each well. A small window time will only account for very discrete levels of fluorescence. These might include sudden spikes of radiation since fluorescence is not a uniform process. Hence we will get variation between samples of equal expression rates. A larger counting time results in a larger window size and hence a more average reading is taken from each sample smoothing out the variation due to the randomness of fluorescence emition.
Care must be taken however because larger counting times will lead to faster fluorescence bleaching. A comprimise between the two must be found.
Protocol | |
Results |
Aims:
Determine the optimum counting time for the fluorometer.
Avoid fluorescence bleaching.
[+] Constant Conditions
[+] Variables
[+] Sampling
[+] Controls