contents

1. Introduction

2. Summary of the Experiment

3. Results

3-1. Plate reader

3-2. Plasmid

5-3. Assay Protocol

4. Discussion

5. Materials and Methods

5-1. Strains

5-2. Plasmid

5-3. Assay Protocol

6. Reference

1. Introduction

　　　　　　

In iGEM 2015, the Interlab Study was held, where we measured the expression level of GFP using three designated devices. It was the first time for our team to join this Interlab Study. In addition to the three designated devices, we also measured the expression level of GFP from a positive control and a negative control using the flow cytometer and the plate reader. Also, for the plate reader, we succeeded in calculating the absolute unit by drawing the calibration curve using sodium fluorescein.

2. Summary of the Experiment

　　　　　　

Our purpose was to obtain the fluorescence data of the three designated devices and to compare them. We prepared Device1〜Device3, Positive control and Negative control as shown below. We measured the exact same colonies of the exact same samples with both the plate reader and the flow cytometer.

Device 1: J23101 + I13504(pSB1C3)

Device 2: J23106 + I13504(pSB1C3)

Device 3: J23117 + I13504(pSB1C3)

Positive control: BBa_I20270(pSB1C3)

Negative control: BBa_R0040(pSB1C3)

Fig.3-7-2-1. designated devices

3. Results

3-1. Plate reader

　　　　　　

First of all, we calibrated our plate reader by confirming the linear relationship between sodium fluorescein concentration and fluorescence (Figure. 3-7-3-1). The way we obtained the calibration curve is descried in the 4. Material and Method section.

Fig.3-7-3-1. Calibration curve

　　　　　　

We measured three colonies(#1〜3) three times (Technical replicate 1〜3) per each sample (Device1〜3, positive control and negative control).
  Using the calibration curve (Figure. 3-7-3-1), we were able to obtain the absolute unit of fluorescence (Table. 3-7-3-1). The way we obtained the absolute unit is described in the 4. Material and Method section. These results show that the intensity of fluorescence was in the following order, Device1>Device2>positive control>Device3>negative control.

Table. 3-7-3-1. The absolute unit of fluorescence intensity

We calculated the arithmetic mean for each sample by adding the nine values of all three colonies and dividing it by 9. We also calculated the standard deviation for each sample from the calculated arithmetic mean. (Table. 3-7-3-2)

Table. 3-7-3-2. Arithmetic mean (Mean) and Standard deviation (S.D) of samples.

4. Discussion

　　　　　　　まだまだいける。

5. Materials and Methods

5-1. Strains

5-2. Plasmids

5-3. Assay Protocol

6. Reference

　　　　　　　めでたし、めでたし。