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 protein expression in E. coli BL21(DE3) cells. Documentation:

The expression system used contained the following parts:

• the BBa_J23110Constitutive promotor
• the BBa_B0034Ribosome binding site

To verify eforRed's absorbance and emission, the construct was expressed in E. coli BL21 (DE3). The bacteria containing the constitutive promotor (pCons) was compared to a negative control (Figure 1). Thereafter the eforRed expressing bacteria was centrifuged, resulting in a pellet with a burgundy color. (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. The culture tubes had a constant supply of oxygen by using cotton plugs which is important for the folding of the eforRed chromophore, therefore leading to increased expression.

Figure 1. The picture to the left depict a E. coli BL21 (DE3) culture expressing pCons-eforRed (left tube) versus a negative control (right tube) with E.coli BL21 (DE3) containing no construct after 48 hours incubation in 37°C. The top right picture displays centrifuged cultures of E. coli BL21 (DE3) with pCons-eforRed in UV-light. The right picture is a culture of E.coli BL21 (DE3) expressing eforRed which has been incubated for 48 hours in 37 degrees Celsius and centrifuged at 12 000g 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) containing pCons-eforRed. An eforRed culture was spread on an LB-agar plate containing 25 µg/ml chloramphenicol. The agar culture were 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. <i> E.coli BL21 (DE3) colonies presented in visual light (right side) and illuminated in 302 nm UV-light (left side). </i>

To test the oxygen dependency of the protein production of eforRed in E.coli BL21 (DE3) , a platereading was conducted. E. coli BL21 (DE3) was grown O.N. in 37 degrees Celsius to an 2.0 OD₆₀₀ and diluted to 0.49 OD₆₀₀, 200 ul of the the bacteria was pipetted into 96-well plates with replicates of 4. Oxygen access was varied by piercing different numbers of holes (0,1,2,3 and 4) in the plastic film of the 96-well plate. The experiment showed that 4 holes in plastic film gave the highest protein yield and that the access to oxygen effects E. colis BL21 (DE3) production of eforRed.

Figure 3. To the left is a of platereading E. coli BL21 (DE3) expressing eforRed with varying access to oxygen. The holes were made in the plastic cover of a 96-well plate and the test was run for 16 hours in 37 degrees Celsius. The plate can be seen on the right pictures left side. The plate to the right is E.coli BL21 (DE3) with a different expression system used to test a strong green/yellow fluorescent protein. Both plates were illuminated in 302 nm UV-light.

A study of the molecular weight of pCons eforRed expressed in E.coli BL21 (DE3) was done by sonicating the cells and performing a SDS-PAGE electrophoresis on the lysate (Figure 4.). Biorads "Precision Plus Protein Dual Color Standards" was used as the protein ladder in the electrophoresis.

Figure 4. SDS-page of sonicated E.coli BL21 (DE3) lysate with pCons-eforRed. Biorads "Precision Plus Protein Dual Color Standards" was used as the protein ladder. The visible band on the gel lies between 25 and 37 kD which corresponds to the molecular weight of eforRed which is 26.1 kDa.

Sequence and Features BBa_K864402 SequenceAndFeatures '''Note:''' This part did not have a reference sequence. A reference sequence has since been added based on the part's documentation; a composite part using the following basic parts: BBa_J23110 - BBa_B0034 - BBa_K592012- iGEM HQ