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

4. Discussion

5. Materials and Methods

5.1. Construction

5.2. Assay Protocol

6. Reference

1. Introduction

　　　　　　

Temperature increase is required in the expression of BBa_K1333309 (J23119_K115002_E1010) constructed by iGEM 2014 SYSU-China. RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. Translation initiating temperature allows the disconnection of the coupling of the hydrogen bonds, which block the SD sequence at low temperature. Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and to initiate translation.
    We improved characterization of BBa_K1333309 by (1) measuring the function of the part at 42 ºC, (2) explicating the way to deal with the 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. 3-7-2-1. Parts that we used

3. Results

　　　　　　

We measured each sample at 30ºC, 37ºC and 42ºC. 37ºC is the translation initiating temperature. Little does the background from the medium affects results for the flow cytometer. Although iGEM 2014 SYSU-China confirmed the function of Pcon_RNA thermometer_rfp at these temperatures, we measured each sample at 42ºC, which is higher than the translation 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. 3-7-3-1).

Fig. 3-7-3-1. 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. 3-7-3-1) 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. 3-7-3-1). 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. 3-7-3-1 The increasing ratios

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

By subtracting the fluorescence intensity of the RNA thermometer_rfp (Fig. 3-7-3-1) from both the fluorescence intensities of Plac_rfp and Pcon_RNA thermometer_rfp (Fig. 3-7-3-1), each at the same temperature, we processed the background derived from the Negative control. We followed the increasing ratios (which backgrounds have been processed) as the section of the standardized fluorescence intensities of RFP, except this time we processed the backgrounds of all the fluorescence intensities. The increasing ratios (which backgrounds have been processed) of the Plac_rfp at 37 ℃ and 42 ℃ show that the fluorescence intensities increased dependent on temperature. We evaluated the increasing ratios (which backgrounds have been processed) of Pcon_RNA thermometer_rfp at 37 ℃ and 42 ℃. Compared to the increasing ratios (which backgrounds have been processed) 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. 3-7-3-2). The increasing ratio (which background has been processed) of Pcon_RNA thermometer_rfp was 2.7 times higher than that of Plac_rfp at 37 ℃. Furthermore, the increasing ratio (which background has been processed) of Pcon_RNA thermometer_rfp was 5.3 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. 3-7-3-2. The increasing ratios (which backgrounds have been processed)

4. Discussion

　　　　　　

We examined that the reason the function of the RNA thermometer was worse at 37 ºC than at 42 ºC was that hydrogen bonds forming RNA thermometer was not enough to change in the structure of the RNA thermometer at 37 ºC.
   Furthermore, there were differences between the increasing ratios and the increasing ratios (which backgrounds have been processed). At 30 ºC, Pcon_RNA thermometer_rfp wasn’t transcribed and did not show expression of RFP. Since the fluorescence intensity of Pcon_RNA thermometer_rfp at 30 ℃ was small, the increasing ratios of between the Pcon_RNA thermometer which background has been processed and the Pcon_RNA thermometer which background has not been processed, greatly differ in both 37 ℃ and 42 ℃(Table. 3-7-4-1). Therefore, it is important to clarify whether the background derived from Negative control was processed or not.

Table. 3-7-4-1. The difference the increasing ratios and the increasing ratios (which backgrounds have been processed)

5. Materials and Methods

5.1. Construction

-Strain

　　　　　　

All the samples were DH5alpha strain.

-Plasmids

　　　　　　

(1) Sample: BBa_K1333309:Pcon_RNA thermometer_rfp(pSB1C3)

Fig. 3-7-5-1.

　　　　　　

(2) Positive control: Plac_rfp_TT(pSB1C3)

Fig. 3-7-5-2.

　　　　　　

(3) Negative Control:RNA thermometer_rfp(pSB1C3)

Fig. 3-7-5-3.

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. Centrifuge the samples at 9000x g, 1 min , 4℃.
5. Remove the supernatants by using P100 pipette and suspend the samples with 1 mL of filtered PBS (phosphate-buffered saline).
6. Dispense all of each suspension into a disposable tube through a cell strainer.
7. 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).

2. SYSU-China 2014