contents

1. Introduction

2. Summary of the Experiment

3. Results

4. Discussion

5. Materials and Methods

5.1. Construction

5.2. Assay Protocol

5.3. Process Data

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: J23119 promoter_RNA thermometer_rfp (BBa_K1333309) (pSB1C3)

Positive control: Plac_rfp_TT(pSB1C3)(BBa_J04450)

Negative control: RNA thermometer_rfp(pSB1C3)

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. 3-8-3-1  RAW data The error bar represents the standard deviation for each sample calculated form the two values of all two colonies. 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)

Fig. 3-8-5-1.

　　　　　　

Positive Control:Plac_rfp_TT(BBa_J04450)(pSB1C3)

Fig. 3-8-6-2.

　　　　　　

Negative Control:RNA thermometer(FourU)_rfp(pSB1C3)

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.

5.3. Process Data

1. Calculated the arithmetic mean of fluorescence intensity of RNAt_rfp at each temperature (30 ºC, 37 ºC and 42 ºC) and used this value as the background derived from Negative control.
2. Subtracted the background derived from Negative control from the fluorescence intensity of Plac_rbs_rfp_tt and Pcon_RNAt_rfp at each temperature.
3. Calculated the rate of increase in fluorescence intensity by normalizing the fluorescence intensity processed the background derived from Negative control of Plac_rbs_rfp_tt and Pcon_RNAt_rfp at 37 ºC and 42 ºC by using the fluorescence intensity processed the background derived from Negative control of Plac_rbs_rfp_tt and Pcon_RNAt_rfp at 30 ºC.
4. Calculated the arithmetic mean of the rate of increase in fluorescence for each sample at 37 ºC and 42 ºC.

6. Reference

　　　　　　

1. Stassen, Oscar MJA, et al., Toward tunable RNA thermo-switches for temperature dependent gene expression. arXiv preprint arXiv:1109.5402 (2011).

2. SYSU-China 2014