RNA thermometer assay

We have characterized previous parts.

1. Introduction

2. Summary of the Experiment

3. Results

4. Discussion

5. Materials and Methods

5.1. Construction

-Strain

All the samples were DH5alpha strain.

-Plasmids

Device 1: J23101 + I13504(pSB1C3)


Fig.3-7-4-1.

Device 2: J23106 + I13504(pSB1C3)


Fig.3-7-4-2.

Device 1: J23117 + I13504(pSB1C3)


Fig.3-7-4-3.

Positive control: BBa_I20270(pSB1C3)


Fig.3-7-4-4.

Negative control: BBa_R0040(pSB1C3)


Fig.3-7-4-5.

-Sequence Data

Please refer to Sequence Data page.

4.2. Instruments and Date

4.2.1. Instruments

-Plate reader

We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJI Film Life Science. The wavelength of light we used to excite the cells was 473 nm. We used BPB1 (530DF20) filter to capture the light emission from the cells. The sampling frequency is only one time.

-Flow cytomerer

We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company. The wavelength of light we used to excite the cells was 488 nm. We used laser detection channel FL1 to capture the light emission from the cells. Laser detection channel Fl1 was used with sensitivity 680 [v]. The sampling frequency is only one time.

4.2.2. Date

Cloning of constructs was confirmed by August 21st 2015. Transformant plates were from 24 August 2015. All the samples were measured on August 27th 2015.

4.3. Protocol

4.3.1. Plate reader

1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3 mL LB medium containing chloramphenicol (35 microg / mL) at 37 °C for 17h and shake at 180 rpm.
2 .Measured the OD590 of each sample and diluted each sample to adjust OD590 within 5% of 0.5.
3. Set the plate reader to measure GFP.
4. Take 1 mL of the samples, and centrifuge at 9000x g, 1 min, 4°C.
5. Remove the supernatants by using P1000 pipette.
6. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.
7. Place 200 μL of each sample into the 96-well plate as described in Table. 3-7-4-1.
8. Measure the fluorescence intensity with plate reader.
9. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.


Table. 3-7-4-1. Position of samples in 96-well plate

4.3.2. Flow cytometer

1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3m LB medium containing chloramphenicol (35 microg / mL) at 37°C for 17h and shake at 180 rpm.
2. Start preparing the flow cytometer 1 h before the end of incubation.
3. 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.
4. Centrifuge the samples at 9000x g, 1min, 4°C.
5. Remove the supernatants by using P1000 pipette and suspend the samples with 1mL 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.

4.4. How to process the data

4.4.1. Plate reader

-How to draw the calibration curve
1. Place 200 μL of various concentrations of sodium fluorescein (500, 375, 250, 125, 50, 25, 10, 5 ng / mL and PBS only) into the 96-well plate in triplicate.
2. Measure the fluorescence intensity with the plate reader.
3. Rotate the 96-well plate 180 degrees horizontally.
4. Measure the fluorescence intensity again.
5. Determine the auto-fluorescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence.
6. Subtract background fluorescence from each fluorescence intensity value of each well containing sodium fluorescein.
7. Take the arithmetic mean of the three technical replicates of sodium fluorescein of each concentration.
8. Draw the calibration curve.

-How to obtain the absolute unit of fluorescence intensity
1. Measure the fluorescence intensity with the plate reader.
2. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.
3. Calculate the arithmetic mean of these two results.
4. Determine the auto-florescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence.
5. Subtract the background fluorescence from each well containing the samples.
6. Divide them by the value of OD590 of each sample.
7. Calculate the ng / mL fluorescence per OD590 unit by the formula we obtained from drawing the calibration curve.

4.4.2. Flow cytometer

Cells were gated according to the side scatter (SSC) and the forward scatter (FCS) to exclude cell debris and impurities.

4.5. Individuals responsible for conducting Interlab study

Misa Minegishi : Measured the devices and processed the data.
   Yuta Yamazaki : Measured the devices and processed the data.
   Hiraku Tokuma : Created the devices.
   Riku Shinohara: Created the devices.

5. Reference

2014 Imperial Interlab Study

Team:Tokyo Tech/Experiment/RNA thermometer assay