How close can the numbers be when fluorescence is measured all around the world ?

Gfp Fluorescence of E.coli

In order to support the interlab approach, our team participate to the Fourth International InterLab Measurement Study. We are glad to contribute to this studie based on reliable and repeatable measurement. The challenge this year consist in etablishing a GFP measurement protocol that can be used produce comparable GFP measurements on plate readers. Furthermore, teams are also going to test some RBS devices that are intended to make gene expression more precise and reliable.

Materials and Methods:

We used plasmids containing different promoter from the 2017 distribution kit and transfected them into E.coli. Before measuring fluorescence expression, we used LUDOX and fluorescein to calibrate the TECAN.

We prepared a dilution series of fluorescein in 4 duplicates and measure the fluorescence in a 96 well plate in a plate reader. By measuring these in all standard modes, we generated a standard curve of fluorescence for fluorescein concentration. We used this to correct cell based readings to an equivalent fluorescein concentration and convert this into a concentration of GFP.

Results

After measuring fluorescence readings and OD, we measured ratio of Fluorescence/OD in order to determine promotor strengh.

Promotor strength:

• TD1,TD4:1791
• TD2,TD5:1185
• TD3,TD6:162

RBS:TD1, TD2, TD3

1>4>2>3>5>6

iGEM Protocol