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Team:NYMU-Taipei/Experiments/Speedy switch
From 2010.igem.org
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(→Reporter Assay1) |
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*fig.1 is the original chart of the experiment.N line and NT line are control.N line stands for the sequence didn't have promoter.NT line stands for the sequence didn't have promoter but added 0.1μM theophylline.N line is compared with 0μM and it shows that even though the sequence didn't have promoter | *fig.1 is the original chart of the experiment.N line and NT line are control.N line stands for the sequence didn't have promoter.NT line stands for the sequence didn't have promoter but added 0.1μM theophylline.N line is compared with 0μM and it shows that even though the sequence didn't have promoter | ||
==Reporter Assay1== | ==Reporter Assay1== | ||
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! !! Theophylline concentration || Theophylline(0.1M) || PFRC liquid(μl)||FRC liquid(μl)|| Cm50(μl) | ! !! Theophylline concentration || Theophylline(0.1M) || PFRC liquid(μl)||FRC liquid(μl)|| Cm50(μl) | ||
Revision as of 15:51, 27 October 2010
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Contents |
Method
- Protocol:
1.Selected genes to be reported are incubated overnight in an LB liquid culture at 37oC and 180-200rpm. This makes sure they are fresh in the morning. Positive and negative controls are also needed.
2.Overnight liquid culture is diluted to OD600 of 0.1, Theophylline is added at concentrations ranging from 0.01mM to 20mM, and the mix incubated for 2-2.5 hours.
3.Measurement of OD at 2 hours: For each used well in the 96-well plate: Take 200uL from the liquid (make sure you pipette this step well) and put it in a cuvette to read the OD600. Note down the OD600 ["OD at 2 hours"], then take the liquid in the cuvette and put it in the right place in the 96-well plate.
4.Measurement of fluorescence: Continuous measurement of fluorescence with the excitation/emission wavelengths 488/511nm for 2 hours, with one fluorescence data point every 2 minutes.
5.Measurement of OD at 4 hours: For each used well in the 96-well plate: Take the liquid from the well and put it in the cuvette to measure the OD ["OD at 4 hours"].
Reporting Assay
- fig.1 is the original chart of the experiment.N line and NT line are control.N line stands for the sequence didn't have promoter.NT line stands for the sequence didn't have promoter but added 0.1μM theophylline.N line is compared with 0μM and it shows that even though the sequence didn't have promoter
Reporter Assay1
| Theophylline concentration | Theophylline(0.1M) | PFRC liquid(μl) | FRC liquid(μl) | Cm50(μl) | |
|---|---|---|---|---|---|
| O | 0 μM | 0 | 261 | 0 | 4 |
| A | 10 μM | 0.4 μl | 261 | 0 | 4 |
| B | 50 μM | 2 μl | 261 | 0 | 4 |
| C | 100 μM | 4 μl | 261 | 0 | 4 |
| D | 200 μM | 8 μl | 261 | 0 | 4 |
| E | 500 μM | 20 μl | 261 | 0 | 4 |
| F | 1000 μM | 40 μpl | 261 | 0 | 4 |
| G | 2000 μM | 80 μl | 261 | 0 | 4 |
| H | 4000 μM | 160 μl | 261 | 0 | 4 |
| I | 8000 μM | 320 μl | 261 | 0 | 4 |
| J | 10000 μM | 400 μl | 261 | 0 | 4 |
| K | 20000 μM | 800 μl | 261 | 0 | 4 |
| NT | 100 μM | 4 μl | 0 | 261 | 4 |
| N | 0 μM | 0 μl | 0 | 261 | 4 |
[fig.1]
.png)
- We test 12 different concentrations of theophylline to induce riboswitch.
[fig.2]
Reporting Assay2
.png)
[fig.3]
- We test 12 different concentrations of theophylline to induce riboswitch.
[fig.4]
Reporting Assay3
.png)
[fig.5]
- We test six different concentration of theophylline to induce riboswitch.
[fig.6]
Repoting Assay4
.png)
[fig.7]
- We test 6 different concentration of theophylline to induce riboswitch.
[fig.8]
