Team:HFLS ZhejiangUnited/InterLab

Realizing the importance of reliable and repeatable measurement for engineering biology, we are excited to participate in the Fifth International InterLaboratory Measurement Study. As a high school team, we choose to participate in “Plate Reader and CFU” Study.

We followed the standard protocol including LUDOX Protocol, Microsphere Protocol, Fluorescein Protocol, Cell Measurement Protocol, and Colony Forming Units Study, etc. (Detailed protocol information can found at https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf). During the whole InterLab study process, everyone, instructed by the team PI, studied the protocol carefully and worked on providing accurate and reliable experiment data to the Measurement Committee.

Materials

Provided in Kit: LUDOX CL-X, Silica beads, Fluorescein, Devices (R0040, I20270, J364000, J364001, J364002, J364007, J364008, J364009)

Provided by ourselves: 96 well plate (black with clear flat bottom preferred), ddH2O, PBS (pH 7.4-7.6), Competent cells (Escherichia coli strain DH5α), LB media, Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH).

Methods

We used a Tecan Infinite M200pro plate reader to measure OD600 and fluorescence values. The excitation wavelength is 485 nm, the Emission wavelength is 530 nm, and the Firmware is V 3.37.

OD600 measurements were taken in Corning 96 well assay plates (black with a clear flat bottom). The experiments performed at the College of Life Science, Zhejiang University, under the supervision of their instructor (Dr. Fangliang Huang, who is also the instructor of 2018 iGEM Team ASTWS-China). We appreciate the use of their plate reader and 96 well assay plates they offered us – see our collaborations page for more details (https://2018.igem.org/Team:HFLS_ZhejiangUnited/Collaborations).

Results:

We submitted the completed spreadsheet and four online forms to the iGEM measurement committee before the deadline (July 27) and received confirmation that the results accepted on the July 31. Calibration

1. OD600 Reference Point

Table 1 Absorbance at 600 nm for LUDOX and H2O.

2. Particle Standard Curve

Figure 2 (a) The normal scale and (b) log scale of particle standard curve.

3. Fluorescein standard curve

Figure 3 (a) The normal scale and (b) log scale of fluorescein standard curve

Cell Measurement

1. Transformation of the devices

In the first day, we transformed the required plasmids (all in pSB1C3) in Escherichia coli strain DH5α, followed is the pictures after incubating overnight at 37 °C.

Figure 4 Devices

2. After transformation, cell growth, sampling, and assay, we collected the raw plate reader measurements including Fluorescence raw readings and Abs600 raw readings. Then the experimental values were gained as follows:

Table 2 Experimental values of fluorescence per OD

Table 3 Experimental values of fluorescence per Particle.

Colony Forming Units per 0.1 OD600 E. coli cultures

Following the protocol provided by iGEM measurement, we accomplished this part and the results are as follows.

Table 4 Colony Forming Units per 0.1 OD600 E. coli cultures

Collaboration

Our interlab study was done in collaboration with two other teams (ASTWS-China, Worldshaper-XSHS). Before beginning the experiment, we carefully studied and discussed the official protocol provided by iGEM measurement together. Worldshaper-XSHS lends us their 96-well plates and LB media when ours are not adequately prepared. Moreover, the experiments performed at the College of Life Science, Zhejiang University, under the supervision of their instructor (Dr. Fangliang Huang, who is also the instructor of 2018 iGEM Team ASTWS-China). We appreciate the use of their plate reader they offered us. On the other hand, we helped worldshaper-XSHS and ASTWS-China analyze the data when their first submission failed.

See our collaborations page for more details (https://2018.igem.org/Team:HFLS_ZhejiangUnited/Collaborations).

Conclusion

Through the InterLab measurement study, we realized that carefulness and patience are extremely important for the experiment. We are very excited when the first submission of our data was confirmed to be accepted. We hope that our work could help advance the development of engineering biology.