Revision as of 01:15, 11 October 2018

As a team, we took part in iGEM’s Fifth International Interlaboratory Study in synthetic biology. Difficulty in taking reliable and reproducible measurements remains a key obstacle in the field of synthetic biology, especially for fluorescence data. Data from different groups usually cannot be compared because they are reported in different units or processed in different ways. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements.

For the fifth installment of the InterLab, iGEM wants to answer the following question: "Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?" The parts used included six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.

Our involvement in the study required that we submit measurement data dealing with the fluorescence of GFP and the OD associated with cells transformed the six different test devices. Throughout our experiments, we tested eight plasmids (two controls and six test devices), and we measured the absorbance and fluorescence of our samples using a Tecan Infinite m1000 pro plate reader. We broke the given procedure into five main components: (1) transforming the two controls and six test devices into competent DH5α cells, (2) measuring the OD600 reference point of LUDOX CL-X, (3) graphing a particle standard curve for monodisperse silica microspheres and a Fluorescein standard curve (4) measuring the GFP fluorescence and absorbance of samples of previously transformed DH5α cells taken over two hour intervals and (5) counting colony forming units (CFUs) per 0.1 OD600 E. coli cultures.

We enjoyed taking part in the interlab study. The instructions/ procedures provided by iGEM were very clear and easy to follow, as a result we did not have to make any modifications. We conducted the procedures as they were given. Before we conducted the fourth and fifth component of interlab we made sure to label and prepare all the materials ahead of time. As a result, we didn't run into any complications and were able to collect all of the data in a timely manner. Since, the experiments are on such a large scale it is important to make sure that you plan ahead.

OD600 Reference Point of LUDOX CL-X

Abs600 and OD600 Data/Calculations for LUDOX-HS40 and Water

Particle Standard Curve

Fluorescein Standard Curve

4. Measurement of Abs600 and Fluorescence of Transformed Cell Samples

Raw Plate Readings for Abs600 and Fluorescence

Fluorescence per OD

Fluorescence per cell

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