1. Interlab Study

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As a first step to contribute to this year’s iGEM competition we decided to participate in iGEM’s fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM’s measurement committee in an effort to establish standardized, reliable and repeatable mea- surement tools for the iGEM community and the synthetic biology community as a whole. Requirement for participating in this year’s study, three essential things were needed. Firstly, we needed access to a plate reader that is qualified to measure GFP fluorescence. In fact, we had access to such a plate reader, namely Tecan Infinite M200 Pro plate reader. Secondly, we needed competent DH5α E.coli which were prepared from glycerol stocks. Lastly, we needed to use the materials iGEM had provided. Combining all of these parts made us ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD₆₀₀ and the fluorescence of the cells carrying our plasmids. The workflow can be separated into four segments. The first segment is the transformation of all the plasmids into competent DH5α cells.

1.1 Transformation of 8 test devices into competent DH5α

The transformation was successful for all plasmids and resulted in a sufficient amount of colonies on all of the plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37 C and 220 rpm.

1.2 Measurement of LUDOX-HS40 OD600 Reference Point

The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD600 measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following table

With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.

1.3 Graphing a fluorescein fluorescence standard curve

In the third segment we were instructed to measure a standard curve for fluores- cence of fluorescein which can be used to correct our cell based readings to an equivalent fluorescein concentration. Following the instructions, we could ob- tain following results for the serial dilution of fluorescein in phosphate buffered saline.

The data give us a more or less linear regression line when plotted in a logarithmic scale but behave more like a quadratic equation when plotted normally.

1.4 Measurement of OD600 and Fluorescence of Transformed Cells

In the last of the four segments the OD600 and the fluorescence of the trans- formed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following tables and calcu- lations which were conducted with the values we obtained from the standard curves and the reference point.

References