contents

1. Introduction

2. Summary of the Experiment

3. Results

3.1. The fluorescence intensities of RFP

3.2. The standardized fluorescence intensities of RFP

3.2.1. The standardized fluorescence intensities of RFP

3.2.2. The standardized fluorescence intensities of RFP after processing the background derived from the Negative control

5.2. Assay Protocol

5.3. Process Data

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_E115002_E1010) constructed by iGEM 2014 SYU-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 initiating temperature, the disconnection of the coupling of the hydrogen bonds, which block the SD sequence, initiates. Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and the transcription of SD sequence is initiated. We improved characterization of BBa_K1333309 by (1) measuring the function of the part at 42 ºC, (2) explicating the way to deal with background derived from Negative control, (3) measuring with the flow cytometer. To express the feeling of the E. coli which fell into dilemma, we measured the temperature dependence of the RNA thermometer.

　　　　　　

We think that this experiment is meets Gold medal criteria.Description

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 the RNA thermometer at 30 ºC, 37ºC and 42 ºC by using the flow cytometer. We prepared the samples as shown below.

Sample: BBa_K1333309:Pcon_RNA thermometer_rfp(pSB1C3)

Positive control: Plac_rfp_TT(pSB1C3)

Negative control: RNA thermometer_rfp(pSB1C3)

Fig. 3-8-2-1. Parts that we used

3. Results

　　　　　　

We measured each sample at 30 ºC, 37 ℃ and 42 ℃. 37 ℃ is the transcription initiating temperature. Although iGEM 2014 SYU-China confirmed the function of Pcon_RNA thermometer_rfp at these temperatures, we measured each sample at 42 ºC, which is higher than the transcription initiating temperature.

3.1. The fluorescence intensities of RFP

　　　　　　

We found that the fluorescence intensities of both Pcon_RNA thermometer_rfp and Plac_rfp increased along with the rise of the temperature (Fig. 3-8-3-1).

Fig.3-8-3-1. RAW data

　　　　　　

The error bar represents the standard deviation for each sample calculated from the two values of all two colonies.

3.2. The standardized fluorescence intensities of RFP

3.2.1. The standardized fluorescence intensities of RFP

We got the increasing ratios of fluorescence intensities at 37 ℃ and at 42 ℃ by dividing the each of the raw fluorescence intensities (Fig. 3-8-3-1) by those at 30℃. The increasing ratios of the Plac_rfp at 37 ℃ and 42 ℃ show that the fluorescence intensities increased dependent on temperature. We evaluated the increasing ratios of Pcon_RNA thermometer_rfp at 37 ℃ and 42 ℃. Compared to the increasing ratios of Plac_rfp, those of the Pcon_RNA thermometer_rfp were higher at respective temperature. We observed not only the increase in the fluorescence intensities dependent on temperature but also the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-8-3-1). The increasing ratio of Pcon_RNA thermometer_rfp was 1.3 times higher than that of Plac_rfp at 37 ℃. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp was 3.2 times higher that of Plac_rfp at 42 ℃. These differences of the increase ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42 ℃ compared to 37 ℃.

Table. 3-8-3-1 The increasing ratios

3.2.2. The standardized fluorescence intensities of RFP after processing the background derived from the Negative control

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