IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1: Difference between revisions
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*Temperature: 25 <sup>o</sup>C (Room temperature) | *Temperature: 25 <sup>o</sup>C (Room temperature) | ||
* | *20 µl aliquots from the commercial cell extract mixture in each well | ||
*20 µl aliquots from the home made cell extract mixture in each well | |||
*20 µl of plasmi DNA in each sample | |||
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Revision as of 16:18, 28 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 | 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
1.2 Initial testing for Infector Detector
Overall Status | |
Construct | Status |
pTet-LuxR-pLux-GFP | INCOMPLETE |
Construct: pTet-LuxR-pLux-GFP
Test to see if construct will express in vivo. Experiments carried out in incubator at 25oC 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 | 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:
- Complete
[+] Constant Conditions
[+] Variables
[+] Sampling
[+] Controls
2.2 Initial testing for Infector Detector
Overall Status | |
Construct | Status |
pTet-LuxR-pLux-GFP | INCOMPLETE |
Construct: pTet-LuxR-pLux-GFP
Protocol | |
Results |
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.
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 infecter detector 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
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
3.2 Investigate the viability of home-made cell extract and reaction buffer together with the commercially bought cell extract and pre-incubation mix
Status | |
Planned for 29/08/2007 | INCOMPLETE |
Construct: pTet-GFP
This experiment is done to test if the commmercial cell extract and pre-incubation mix, if mixed in a ratio of 1:1 with the home made cell extract and reaction buffer, will produce sufficiently satisfying results to be used for carrying out the rest of the experiments for CELL BY DATE and INFECTOR DETECTOR.
Protocol | |
Results |
Aims:
To determine the working efficiency of the mixture of commercial and home-made cell extracts.
[+] Constant Conditions
[+] Variables
[+] Sampling
[+] Controls