Difference between revisions of "Part:BBa K864402"

Revision as of 11:15, 12 October 2019

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 (BBa_K864402) was expressed in e.Coli BL21 (DE3). The bacteria containing the constitutive promotor (p.Cons) was compared to a negative control, (Figure x, left side). Thereafter the eforRed bacteria was centrifuged which displayed a pink pellet (Figure x, top right corner). To demonstrate the fluorescence of eforRed, the pellet was placed on a UV-table emitting a wavelength of 302 nm, (Figure X, down-right corner), which displayed a pink glowing pellet and veried the fluorescent of eforRed.

T--Linkoping Sweden--eforred-pellet-photo.png

Figure 1. To the left This biobrick after 48 hours in 37 degrees Celsius (both pictures). To the left is cultured E. coli BL21 (DE3) Gold cells with this biobrick in white light. The culture tubes had a constant supply of oxygen (cotton plugs) which is important for the chromophore/fluorophore of eforRed to develop. In the bottom right a culture of BL21 (DE3) Gold which has been incubated for 48 hours in 37 degrees Celsius has been centrifuged at 12 000 g for 10 min. The result is a pellet of eforRed expressing bacteria with a burgundy color. The same pellet in the top right was put on a UV table (302 nm) which resulted in a pink glowing pellet.

To test the fluorescence, e.Coli BL21(DE3) containing pCons eforRed were spread on a agar plate containing 25 µg/ml chloramphenicol. This was done to illustrate the ability of eforRed to emit strong fluorescens on an agar plate in UV-light(SKRIVA MERA SOM EN METOD)??, and at the same time have a clear Burgundy color in visible light on the plate.

T--Linkoping Sweden--eforred-agar-photo.png

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.

To test the affect of oxygen supply on the recombinant protein production of eforRed in e.Coli BL21 (DE3) a platereading was conducted. The oxygen supply was varied by sticking different amount of holes in the plastic film of a 96-well plate. The experiment showed that the access to oxygen affects e.Colis recombinant protein production of eforred in a 96 well plate.

T--Linkoping Sweden--eforredvsmngphoto.jpeg

Figure 3. To the left is a spectrophotometric experiment where varying levels of oxygen supply were tested. The holes were made in the plastic cover of a 96-well plate and run for 16 hours in 37 degrees Celsius, this results shows the oxygen dependency of eforReds chromophore/fluorophore development. To the right is this biobrick in a 96-well plate, the culture has been in the well O.N in 37 degrees Celsius with varying levels of oxygen supply. The plate to the right is different expression systems used to test a strong green/yellow fluorescent protein. These results show eforReds ability to fluorescence, making it a fluorescent as well as a chromoprotein. 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) was done by sonicating the E.coli cells containing p.Cons-eforRed and then performing a SDS-PAGE Electrophoresis on the lysate (Figure z.).

Figure 4.SDS-page of the sonicated BL21(DE3) lysate with 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.

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

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+A study of the molecular weight of p.Cons-eforRed expressed in E.coli BL21(DE3) was done by sonicating the E.coli cells containing p.Cons-eforRed and then performing a SDS-PAGE Electrophoresis on the lysate (Figure z.).<br><br>
 </html>[[Image:T--Linkoping_Sweden--eforred_SDS-page.png|100px|thumb|left|<b><i>Figure 4.</i></b>SDS-page of the sonicated BL21(DE3) lysate with 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.]] <html></html>
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