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.

For more details on the procotol : iGEM Protocol

Results

After measuring fluorescence readings and OD, we calculed ratio of Fluorescence/OD in order to determine promotor strengh. Device 1 and device 4 containing promoter J23101 had high fluorescence production. On contrary, device 3 and device 6 containing promoter J23117 maked a low fluorescence production. Device 2 and device 5 containing promoter J23106 was observed to have a media increase of fluorescence production. This results are suggesting that the promoter J23101 had the strongest affinity for initial RNA polymerase binding comparing to other devices promoter J23117 and J23106.

Promotor strength:

• J23101(TD1,TD4):1791
• J23106(TD2,TD5):1185
• J23117(TD3,TD6):162