Interlab

Introduction.

Reliable results are crucial in science in general, thus also in synthetic biology. As so, it is important to establish robust protocols which lead to similar outcomes independent from the lab they are generated in. Therefore, iGEM’s Measurement Committee has been developing a robust procedure for measuring green fluorescent protein (GFP), an intensively used marker in synthetic biology.
To make our contribution we decided to take part in this year’s 4^th International InterLab Measurement Study organized by iGEM. The aim of the study is to test whether it is possible to obtain reproducible results for fluorescence measurements carried out all around the world in different labs, with different instruments, by different people using the exact same afore mentioned protocol.
For this year’s study 6 different GFP-expression plasmids, containing divergently strong constitutive promoters, as well as a positive and a negative control were investigated. Therefore, an OD₆₀₀ reference point and a fluorescein fluorescence standard curve were generated, before the constructs were transformed into E. coli DH5α and expression levels were determined by OD₆₀₀ and fluorescence measurement in a plate reader in 2 hour intervals for a total of 6 hours.
For further information visit the Interlab website.

Material and Methods.

Material

• InterLab Parts and Measurement Kit
• Plate reader: Tecan Infinite^® M200
• Bacteria: E. coli DH5α

Methods

• The transformations were carried out according to our Transformation Protocol
• The measurement study was carried out according to iGEM's InterLab Plate Reader Protocol

Results.

OD₆₀₀ reference point

The determination of the OD₆₀₀ reference point addresses two major issues: transforming absorbance measurements into OD₆₀₀ measurements and generating instrument independent data. LUDOX-S40 is used as a single point reference to obtain the conversion factor, which is calculated by dividing the Reference OD₆₀₀ by the determined absorbance. The results of our measurements are shown in table 1.

Table 1: Measurements for the OD₆₀₀ reference point - a conversion factor to transform absorbance data into a standard OD₆₀₀ measurement.

Fluorescein standard curve

A fluorescein fluorescence standard curve is generated to be able to transform cell based readings into equivalent fluorescein concentrations, what can then be converted into GFP concentrations. Our fluorescein standard curve is shown in figure 1.

Figure 1: Fluorescein fluorescence standard curve: linear scale (left) and log scale (right).

Raw data of plate reader measurement and final results

After transforming E. coli DH5α with the different plasmids, two colonies from each plate are picked and precultures are generated. The next day OD₆₀₀ of all cultures is measured and they all are brought to a similar OD₆₀₀. Afterwards the cultures are incubated again and samples are taken every 2 hours for an overall period of 6 hours. Finally, absorbance and fluorescence of all samples is determined in the plate reader.
Our raw data and final results are displayed in table 2 and 3.

Table 2: Raw data of plate reader measurement.

Table 3: Final results of fluorescence and OD₆₀₀ measurement.

Team:BOKU-Vienna/InterLab