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 and then measured the absorbance of the cells with a plate reader (spectrophotometer).
Documentation:

The expression system used contained the following parts:

• the BBa_B0034 ribosome binding site
• the BBa_J23110 Constitutive promotor

To verify eforRed's absorbance and emission, the construct was expressed in E.coli BL21 (DE3) Gold cells. 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 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 E.coli BL21 (DE3) gold cell culture expressing pCons-eforRed (left tube) versus a negative control (right tube) with E.coli BL21 (DE3) gold cells containing no construct after 48 hours incubation in 37°C. The top right picture displays centrifuged cultures of E.coli BL21 (DE3) gold cells with pCons eforRed in UV-light. 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. The right picture is a culture of BL21 (DE3) gold cells expressing 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 is presented in visual light (right side) and illuminated in 302 nm UV-light (left side).

To test the oxygen dependency of the protein production of eforRed in E.coli BL21 (DE3) gold cells, a platereading was conducted. The oxygen access was varied by piercing different numbers of holes in the plastic film of the 96-well plate. The experiment showed that the access to oxygen effects E.colis protein production of eforRed.

Figure 3. To the left is a platereading E.coli BL21 (DE3) gold cells protein production of 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 results shows that the production of eforRed was effected by oxygen access. The plate can be seen on the right pictures left side. The plate to the right is E.coli BL21 (DE3) gold cells 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 p.Cons-eforRed expressed in E.coli BL21 (DE3) gold cells was done by sonicating the cells and then performing a SDS-PAGE electrophoresis on the lysate (Figure 4.) with Biorads "Precision Plus Protein Dual Color Standards" as the protein ladder.

Figure 4. SDS-page of sonicated e.Coli BL21(DE3) lysate with p.Cons-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 correspond to the molecular weight of eforRed which is 26.1 kDa.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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: No part name specified with partinfo tag. - BBa_B0034 - BBa_K592012- iGEM HQ