Team:Tokyo Tech/Experiment/RNA thermometer assay
RNA thermometer assay
We have characterized previous parts.
contents
1. Introduction
2. Summary of the Experiment
3. Results
4. Discussion
5. Materials and Methods
5.1. Construction
5.2. Assay Protocol
6. Reference
1. Introduction
ここにコピペ。
2. Summary of the Experiment
ここにコピペ。
3. Results
ここにコピペ。
4. Discussion
ここにコピペ。
5. Materials and Methods
5.1. Construction
-Strain
All the samples were DH5alpha strain.
-Plasmids
Sample:J23119 promoter_RNA thermometer(FourU)_RFP(BBa_K1333309)(pSB1C3)
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Fig. 3-8-5-1. |
Positive Control:Plac_RFP_TT(BBa_J04450)(pSB1C3)
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Fig. 3-8-5-2. |
Negative Control:RNA thermometer(FourU)_RFP(pSB1C3)
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Fig. 3-8-5-3. |
5.2. Assay Protocol
1. Prepare 2 over night cultures for each sample in 3 mL LB medium containing chloramphenicol (25 microg / mL) at 37 ºC for 12 h.
2. Dilute the overnight cultures to 1/100 in fresh LB medium (3 mL) containing chloramphenicol (25 microg / mL) (fresh culture).
3. Incubate the fresh cultures at 37℃ for 8 h.
4. Start preparing the flow cytometer 1 h before the end of incubation.
5. Measure the OD590 and adjust the volume of each sample to centrifuge so that the amount of pellet will be about the same for every sample.
6. Centrifuge the samples at 9000x g, 1 min , 4℃.
7. Remove the supernatants by using P100 pipette and suspend the samples with 1 mL of filtered PBS (phosphate-buffered saline).
8. Dispense all of each suspension into a disposable tube through a cell strainer.
9. Measure fluorescence intensity with flow cytometer.
6.. Reference
1. Stassen, Oscar MJA, et al., Toward tunable RNA thermo-switches for temperature dependent gene expression. arXiv preprint arXiv:1109.5402 (2011).