Part:BBa_K864402

J23110-B0034-eforRed

eforRed eforRed is previously described as BBa_K592012. We submitted a functionally active variant with J23110 and B0034.

Contribution

Group: Linkoping_Sweden iGEM 2019
Author: Andreas Holmqvist and Leo Juhlin
Summary: In this contribution we characterized the visual absorbance and the fluorescence of this construct. We also tested the oxygen dependency of the chromophore protein expression in E.coli BL21(DE3) cells and then measured the absorbance of the cells with a plate reader.
Documentation:

The expression system used contained the following parts:

• the BBa_B0034 ribosome binding site
• the BBa_J23110 Constitutive promotor

To verify eforReds absorbance and emission, the construct was expressed in E.coli BL21 (DE3) gold cells. The bacteria containing the constitutive promotor (p.Cons) was compared to a negative control (Figure 1, left side). Thereafter the eforRed bacteria was centrifuged which displayed a pink pellet (Figure 1, top-right corner). To demonstrate the fluorescence of eforRed, the pellet was placed on a UV-table emitting a wavelength of 302 nm, (Figure 1, down-right corner), which exhibited a pink glowing colour.

Figure 1. The picture to the left depict a cell culture of E.coli BL21 (DE3) gold cell p.Cons eforRed (left tube) versus a negative control (right tube) with E.coli BL21 (DE3) gold cells after 48 hours incubation in 37°C. The top right picture displays a centrifuged cultures of E.coli BL21 (DE3) gold cells with p.Cons eforRed in UV-light. The culture tubes had a constant supply of oxygen with the use cotton plugs which is important for the folding of the chromophore/fluorophore of eforRed. The right picture is a culture of BL21 (DE3) gold cells containing eforRed which has been incubated for 48 hours in 37 degrees Celsius and centrifuged at 12 000 g for 10 min. The result is a pellet of eforRed expressing bacteria with a Burgundy color (bottom right). The same pellet in the top right was put on a UV table (302 nm) which resulted in a pink glowing pellet (top right).

Further characterization was performed in order to demonstrate the absorbance and fluorescence of E.coli BL21 (DE3) gold cells containing p.Cons eforRed were spread on an agar plate containing 25 µg/ml chloramphenicol. The plate was then photographed in visual light and on a UV-table emitting 302 nm (Figure 2 ). The results were the same as above, in visual light (Figure 2, right) the cultures had a burgundy color and on the UV-table the bacteria exhibited a pink glowing colour ( Figure 2, left).

Figure 2. Colonies in the same host as previously in figure 1 is illuminated in 302 nm UV-light, showing eforReds ability to also emit strong fluorescence.