Team:ECUST/InterLab

Interlab

Introduction

As one of the most important measurement marker in synthetic biology,however, the fluorescence data of GFP usually cannot be compared because it has been reported in different units or because different groups process data in different ways.

The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab.

The goal for the fifth interlab is to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD.

Method

First, make three sets of unit calibration measurements: an OD600 reference point, a particlestandard curve, and a fluorescein standard curve.

Secondly, perform the cell measurements.

Finally, fill in the experimental data as required.

Click here for detailed information.

Results

Standard Curve

Figure1.1 OD600 reference point tab
Figure1.1 OD600 reference point tab
Figure 1.2 particle stanard cure
Figure 1.2 particle stanard cure
Figure 1.3 particle stanard cure(log scale)
Figure 1.3 particle standard curve(log scale)
Figure 1.4 fluorescein standard curve
Figure 1.4 fluorescein standard curve
Figure 1.5 fluorescein standard curve (log csale)
Figure 1.5 fluorescein standard curve (log csale)

Measurement

Figure 2.1 Fluorescence Raw Reading
Figure 2.1 Fluorescence Raw Reading
Figure 2.2 Abs600 Raw Reading
Figure 2.2 Abs600 Raw Reading
Figure2.3.a Positive control
Figure2.3.a Positive control
Figure2.3.a Positive control
Figure2.3.b Negative control
Figure2.3.a Positive control
Figure2.3.c Device 1
Figure2.3.a Positive control
Figure2.3.d Device 2
Figure2.3.a Positive control
Figure2.3.e Device 3
Figure2.3.a Positive control
Figure2.3.f Device 4
Figure2.3.a Positive control
Figure2.3.g Device 5
Figure2.3.a Positive control
Figure2.3.h Device 6

Fluorescence per colony

Figure 3.1 Fluorescence per OD at 0h
Figure 3.1 Fluorescence per OD at 0h
Figure 3.2 Fluorescence per OD at 6h
Figure 3.2 Fluorescence per OD at 6h
Figure 3.3 Fluorescence per Particle at 0h
Figure 3.3 Fluorescence per Particle at 0h
Figure 3.4 Fluorescence per Particle at 6h
Figure 3.4 Fluorescence per Particle at 6h

Discussion

Highest average fluorescence was obtained from test device 5, closely followed by device 4 and test device 1.Test device 6 and test device 2 show a modest fluorescence intensity, while test devices 3 have any fluorescence signal as well as the negative group. The order of the promoters given by igem official website is, from strong to weak.test device 4,test device5,test deviece1,test device2,test device6,and test device3. The experimental value is in line with the expected value.

Comparing the fluorescence values at 0h and 6h, it was found that the fluorescence value at 6h did not increase significantly. According to the given protocol from iGEM, the 0h bacterial liquid is not directly measured by fluorescence, but is measured after 6 hours of ice bath. We believe that the ice bath is not conducive to bacterial growth, so a large amount of fluorescent protein is expressed. The experiment confirmed our conjecture. If the 0h bacteria solution is directly measured instead of ice bath, the fluorescence value will be significantly lower than the fluorescence value of 6h.