InterLab

The most direct aim of the entire InterLab is to standardize the fluorescence measurements. And the topic of standardization varies every year to be more specific and comprehensive.

Standardization

Standardization plays an extremely important role in science experiments. There are multiple kinds of methods which will keep the lab results accurate.

1. Every protocol is read and followed carefully by group members and checked by each member.

2. Any indices should be the same unit and same figure.

3. Any experiment is better to hold two control groups (one is 100% source, the other is 100% solution).

4. An ideal standardization experiment needs more than 3 repeating experiments.

Standardization is the process of developing and implementing technical standards. It can help to maximize compatibility, interoperability, safety, repeatability, or quality as well.

Goals

As a whole, the InterLab is trying to minimize variability across labs. Trying to further contribute amenity to the synthetic biological study, instead of measuring one fluorescent molecule, this year the IGEM teams are standardizing the optical measurement of the entire population of GFP (Green Fluorescent Protein) cells. Because ordinarily means to measure populated cells in optical devices have complicated process and uncertain results, this year, we are using massive data to verify if there is a method that can simplify the process and increase comparability.

The method that is been proving by teams is the one that if we can reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD.

Experiment Process

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. Excitation wavelength is 485 nm, Emission wavelength is 530 nm, and the Firmware is V 3.37. OD600 measurements were taken in Corning 96 well assay plates (black with clear flat bottom). The experiments were performed at the College of Life Science, Zhejiang University, under supervision of our instructor. We followed the standard protocols including LUDOX Protocol, Microsphere Protocol, Fluorescein Protocol, Cell Measurement Protocol and Colony Forming Units Study, etc (Detailed protocol information can be found here). 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.

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 were accepted on the August 16.

Calibration

1. OD600 Reference Point

2. Particle Standard Curve

3. 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.

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:

Colony Forming Units per 0.1 OD600 E. coli cultures

Following to the protocol provided by iGEM measurement, we accomplished this part and the result is as follows.

Collaboration

Our interlab study was done in collaboration with two other teams (HFLS_ZhejiangUnited, 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 when ours are not adequately prepared. Moreover, when our first submission was failed, we together with worldshaper-XSHS and HFLS_ZhejiangUnited to analyze the data and find what is the problem is. Thank you for their help given to us. On the other hand, we offered the plate reader for them to do the InterLab study.
Click our collaborations page for more details.

Conclusion

To sum up, the result of this InterLab Study is quite satisfying. All the related replicates shared the similar curve（posted in the previous section）and we were able to figure out that the number of each colony was positively related to the corresponding absorbance. So we roughly concluded that it is possible to simplify the original process with calculated formula（the transition from Absorbance to colony amount）. We are honored to participate in this session of InterLab. We hope that our work could help advance the development of engineering biology.

Team:ASTWS-China/InterLab