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
Transcription of BBa_K1333309 (J23119_K115002_E1010) created by the 2014 iGEM team, SYSU-China, is initiated at 37 ºC. RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. At the transcription initiation temperature, hydrogen bond that block the SD sequence are cut. Therefore, transcription of SD sequence initiates. We improved the description of characterization of BBa_K13333-09 by (1) measuring with the flow cytometer, by ,(2) explicating the way to deal with background derived from Negative control and by (3) measuring each sample cultured at 42 ºC. To represent the feeling of the E. coli which fell into dilemma, we measured the temperature dependence of RNA thermometers.
2. Summary of the Experiment
Our purpose is to confirm the behavior of the RNA thermometer by setting Positive control and Negative control and to characterize the temperature dependence of RNA thermometer at 30 ºC, 37ºC and 42 ºC by using flow cytometer. We prepared samples as shown below.
Sample: J23101 + I13504(pSB1C3)
Positive control: J23106 + I13504(pSB1C3)
Negative control: J23117 + I13504(pSB1C3)
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Fig.3-7-2-1. designated devices |
3. Results
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4. Discussion
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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).