Team:AHUT China/Overview

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     Introduction

We took part in the Fifth International InterLab Measurement Study which ains to achieve the purpose of comparative measurement. The goal of this study is to obtain large amounts of data from labs across the world,to develop absolute units for measurements GFP in a plate reader to eliminate variation between labs.


     Materials

Plate reader: Synergy H1 (Biotek)
Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)
Cell culture shaker: ZWYR-200D

Devices:
Negative control ：BBa_R0040
Positive control ：BBa_I20270
Device 1： BBa_J364000
Device 2： BBa_J364001
Device 3： BBa_J364002
Device 4： BBa_J364007
Device 5： BBa_J364008
Device 6： BBa_J364009
Note: for Device 5, we have not transformed it into DH5⍺ competent cells successfully for many times, therefore, we thank IGEM team of Nanjing University for providing the Device 5.
Calibration material: Provided in the 2018 IGEM distribution kit
Microorganism: Escherichia coli DH5⍺ strains


     Methods

Following iGEM requirements, Team AHUT_China performed measurements according to these 2018 InterLab Protocols https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf


     Results

1.OD 600 reference point

Using OD 600 and H2O to generate the conversion factor for the transformation later. The average of OD600 is 0.063; the correction factor (OD600/ABS600) is 3.500



Fig. 1 LUDOX correct value

2.Particle standard curve

We obtained the two Particle Standard Curve (normal and log scale).



Fig. 2 Particle Standard Curve

Fig. 3 Particle Standard Curve (log scale)

3.Fluorescein standard curve

Dilution serious of fluorescein were prepared and measured in a 96 well plate. A standard curve is generated to correct the cell based readings to an equivalent fluorescein concentration.
We obtained the two Fluorescein Standard Curve (normal and log scale).



Fig. 4 Fluorescein Standard Curve

Fig. 5 Fluorescein Standard Curve (log scale)

4.Cell measurements

 



Fig. 6 Fluorescence Measurements Curve

Test devices 1 and 4 show high fluorescence intensity. Test devices 2 show a modest fluorescence intensity alone with positive control group, while devices3,5,6 barely show low fluorescence intensity alone with the negative control group.



Fig. 7 Raw OD600 Curve

5.We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures



Fig. 8 CFU Result

 

Discussion

For Figure 3, the log graph isn’t a straight line but not 1:1 slope. In figure 6, highest fluorescence was obtained from device 4, closely followed by test device 1. Test device 2 and positive control group show a modest fluorescence intensity and device 5,6 show low fluorescence intensity, while test devices 3 barely have any fluorescence signal as well as the negative group.

Conclusion

It was certainly a technical challenge to Participate in the InterLab Study. Performing the prescribed protocols with adherence to all the InterLab guidelines yielded parts of expected results, and with the completed InterLab Google Forms, confirms our team participation in this InterLab Study.