Team:Nanjing NFLS/InterLab

Team:Nanjing_NFLS 2017-igem.org

InterLab.

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Overview

Experiment summary & Results

Overview

Team Nanjing_NFLS used E.coli DH5alpha for measurements in our InterLab study. The plate reader we used is Biotek Synergy HT, which could measure both absorbance and fluorescence from top of the plate. It has the pathlength correction that could be disabled during the measurement. With this instrument, we successfully completed the measurements in Plate Reader and CFU Protocol for 2018 InterLab study.

Experiment summary & Results

Calibration 1: OD600 Reference point - LUDOX Protocol

For the first calibration, we used LUDOX CL-X as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. This is a necessary conversation as plate reader measurements of absorbance are volume dependent, and the path length defined by depth of the fluid in the wells can vary slightly from well to well.

Here are the results we obtained from this experiment:

Fig. 2.1

Calibration 2: Particle Standard Curve - Microsphere Protocol

For the second calibration, we prepared a dilution series of monodisperse silica microspheres and measure the Abs 600 in your plate reader. This measurement allowed us to construct a standard curve of particle concentration that can be used to convert Abs600 measurements to an estimated number of cells.

Here are the results we obtained from this experiment:

Fig. 2.2-1

Fig. 2.2-2

Fig. 2.2-3

Calibration 3: Fluorescence standard curve - Fluorescein Protocol  

It is necessary for each team to create a standard fluorescence curve in order to compare fluorescence output of test devices between teams. For the third calibration, we prepared a dilution series of fluorescein in four replicates and measured the fluorescence in plate reader. With such measurements, we could construct a standard curve of fluorescence, and were able to use it to convert our cell-based readings to an equivalent fluorescein concentration.  

Here are the results we obtained from this experiment:

Fig. 2.3-1

Fig. 2.3-2

Fig. 2.3-3

Cell measurement

After completing three calibration measurements, we started to perform cell measurements. We used E.coli K-12 DH5alpha to ensure consistency and reproducibility. We used the same plates and volumes that we used in the calibration protocol, as well as the same settings (e.g., filters or excitation and emission wavelengths) that we used in previous calibration measurements.

Here are the results we obtained from this experiment:

Fig. 2.4-1

Fig. 2.4-2

Conclusion & Problems analysis

We are very pleased to participate InterLab study this year. Since it’s the first time for most students in our team to participate in InterLab study, we were at first quite unfamiliar with the goal and process of conducting such experiments. After thoroughly studying the protocols provided by iGEM Measurement Committee, we tried our best to perform the experiments accordingly. However, due to time limits and other conditions, some of our procedures for transfection and competent cell preparation might be a little different from the official protocols, thus might have led to few inaccuracy of the data. We would like to pay more attention to the details in the procedures in the future and what we have learned from InterLab study would certainly help us better arrange the timeline and increase the accuracy in experiments..

We also want to express our sincere thanks to our advisor Mr. Jian Wu for helping us with instruments manipulation and all the other problems occurred during experiments.