RNA thermometer assay

1. Introduction

Temperature increase is required for an RNA thermometer in the enhanced expression of BBa_K1333309 (J23119_K115002_E1010) constructed by iGEM 2014 SYSU-China. The 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 42ºC, (2) explicating the way to deal with the background derived from Negative control, (3) measuring with the flow cytometer.

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

BBa_K1333309: Pcon_RNA thermometer_rfp (pSB1C3)

Positive control: Plac_rfp_TT (pSB1C3)

Negative control: RNA thermometer_rfp (pSB1C3) (Promoter-less control)


Fig. 3-7-2-1. Parts that we used

3. Results

We measured each sample at 30ºC, 37ºC and 42ºC. The translation initiating temperature is 37ºC. Little background from medium affect results for flow cytometer. Although iGEM 2014 SYSU-China confirmed the function of Pcon_RNA thermometer_rfp at these temperatures, we additionally 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 of two samples which derived from two different colonies, respectively.

3.2. The standardized fluorescence intensities of RFP

3.2.1. The standardized fluorescence intensities of RFP

We obtained increasing ratios of fluorescence intensities at 37ºC and at 42ºC by dividing the each of the raw fluorescence intensities (Fig. 3-7-3-1) by those at 30ºC. The increasing ratios of the Plac_rfp at 37ºC and 42ºC show that the fluorescence intensities, even without the RNA thermometer, increased dependent on temperature. We further evaluated the increasing ratios of Pcon_RNA thermometer_rfp at 37ºC and 42ºC. Compared to the increasing ratios of Plac_rfp, those of the Pcon_RNA thermometer_rfp were higher at respective temperatures. This comparison shows not only the increase in the fluorescence intensities dependent on temperature, (Fig. 3-7-3-1) 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 37ºC. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp was 3.2 times higher than that of Plac_rfp at 42ºC. These differences of the increasing ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.

Table. 3-7-3-1. The increasing ratios

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

In this section, we obtained processed fluorescence intensities by subtracting the fluorescence intensity of a negative control, 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 then obtained increasing ratios with background subtraction at 37ºC and at 42ºC by dividing the each of the processed fluorescence intensities by those at 30ºC (Table. 3-7-3-2). The increasing ratios of the Plac_rfp with background subtraction at 37ºC and at 42ºC show that the fluorescence intensities increased dependently on temperature. We evaluated the increasing ratios of Pcon_RNA thermometer_rfp with background subtraction at 37ºC and at 42ºC. Compared to the increasing ratios of Plac_rfp with background subtraction, those of the Pcon_RNA thermometer_rfp were higher at respective temperatures. We observed again the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-7-3-2). The increasing ratio of Pcon_RNA thermometer_rfp at 37ºC with background subtraction was 2.6 times higher than that of Plac_rfp at 37ºC. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp with background subtraction was 6.8 times higher than that of Plac_rfp at 42ºC. These differences in the increasing ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.

Table. 3-7-3-2. The increasing ratios with background subtraction

4. Discussion

We examined that the reason the function of the RNA thermometer was worse at 37ºC than at 42ºC was that the amount of hydrogen bonds forming RNA thermometer was not enough to change in the structure of the RNA thermometer at 37ºC.
Furthermore, differences between the increasing ratios with and without background subtraction disclose the importance of background treatment. At 30ºC, Pcon_RNA thermometer_rfp wasn’t translated enough and showed little expression of RFP. Since the fluorescence intensity of Pcon_RNA thermometer_rfp at 30ºC was small, the increasing ratios of the Pcon_RNA thermometer with and without background subtraction greatly differ in both 37ºC and 42ºC (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 with and without background subtraction

5. Materials and Methods

5.1. Construction

-Strain

All the samples were DH5alpha strain.

-Plasmids

(1)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 37ºC for 12 h.
2. Dilute the overnight cultures to 1/100 in fresh LB medium (3 mL) containing chloramphenicol (25 microg / mL ) in triplicate (fresh culture).
3. Incubate the triplicated fresh cultures each at 30ºC, 37ºC and 42ºC for each sample for 8 h.
4. Centrifuge the samples at 9000x g, 1 min, 4ºC.
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

Team:Tokyo Tech/Experiment/RNA thermometer assay

RNA thermometer assay

contents

1. Introduction

2. Summary of the Experiment