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. Process the data
6. Materials and Methods
6.1. Construction
6.2. Assay Protocol
7. 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: J23119 promoter_RNA thermometer_rfp (BBa_K1333309) (pSB1C3)
Positive control: Plac_rfp_TT(pSB1C3)(BBa_J04450)
Negative control: RNA thermometer_rfp(pSB1C3)
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Fig. 3-8-2-1. Parts that we used |
3. Results
We cultured each sample at 30 ºC that is lower than the transcription initiation temperature and 37 ºC that is higher than the transcription temperature. SYSU-China 14 confirmed the function of J23119 promoter_RNA thermomer_rfp (BBa_K1333309) at these temperatures. In addition, we cultured each sample at 42 ºC that is higher than the transcription initiation temperature. The wavelength of light we used to excite the cells was 642 nm. We used laser detection channel FL3 to capture the light emission from the cells.
We found that the fluorescence intensity of both J23119 promoter_RNA thermometer_rfp (BBa_K1333309) and Plac__rfp_TT (BBa_Jj04450) elevated with the temperature rose (Fig. 3-8-3-1).
Fig. 3-8-2-1. Parts that we used |
4. Discussion
We believed that the reason why the function of RNA thermometer was worse at 37 ºC than at 42 ºC was that hydrogen bond forming RNA thermometer weren’t cut completely at 37 ºC.
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. 3-8-6-2. |
Negative Control:RNA thermometer(FourU)_rfp(pSB1C3)
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Fig. 3-8-5-3. |
6.2. 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).