Difference between revisions of "Part:BBa K864402"
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</html>[[Image:T--Linkoping_Sweden--eforred-agar-photo.png|700px|thumb|left|<div class="figurtext"style=font-size:80%;><i><b>Figure 2.</b> E. coli BL21 (DE3) colonies presented in visual light (right side) and illuminated in 302 nm UV-light (left side). </i>]]<html>
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</html>[[Image:T--Linkoping_Sweden--eforred-agar-photo.png|700px|left|]]<div class="figurtext"style=font-size:90%;><b>Figure 2.</b> Colonies in the same host as previously is presented in white light (right side) and in 302 nm UV-light (left side).<html>
 
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To test the oxygen dependency of the protein production of eforRed in <i>E. coli</i> BL21 (DE3) , a platereading was conducted. <i>E. coli</i> BL21 (DE3) was grown O.N. in 37 degrees Celsius to an 2.0 OD<sub>600</sub> and diluted to 0.49 OD<sub>600</sub>, 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 <I>E. colis</I> BL21 (DE3) production of eforRed.</p>
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To test the oxygen dependency of the protein production of eforRed in BL21 (DE3), the bacteria containing pCons-eforRed was grown O.N. to 2 OD<sub>600</sub> and diluted to 0.49 OD<sub>600</sub> with LB-miller. The bacteria was placed in a 96-well plate in replicates of 4 with 200 µL in each well. The 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. A spectrometry experiment was conducted measuring the fluorescence (excitation 589, emission 609) in 37 °C for 24 hours and the experiment (Figure 3) showed that the access to oxygen effects the folding of eforRed and that 4 holes gave the highest yield.</p>
 
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Revision as of 15:34, 20 October 2019

J23110-B0034-eforRed

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


Eforred plate small.jpg UUChromo.jpg



Usage and Biology

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. The molecular weight of eforRed was also examined with a SDS-PAGE. Documentation:

The expression system used contained the following parts:

To verify eforReds absorbance and emission, the construct was heat shocked into Escherichia coli, using the strain BL21 (DE3), and grown in a Falcon tube O.N. in 37 °C at 25 µg/mL chloramphenicol. Cotton plugs was used as corks for the Falcon tube. The bacterial solution was compared to a negative control with only BL21 (DE3)(Figure 1, left side) which showed the red color of eforRed in a bacterial solution. Thereafter, the eforRed expressing bacteria was centrifuged at 12 000 g for 10 minutes which displayed a burgundy colour (Figure 1, top-right corner). The pellet was also placed on an UV-table emitting light at 302 nm (Figure 1, down-right corner) and exhibited a pink glowing colour. These experiments verifies eforRed fluorescent effect as well as its absorbance in white light.



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

Figure 1. The picture to the left depicts a cell culture of BL21 (DE3) pCons-eforRed (left tube) versus a negative control (right tube) with BL21 (DE3). The top right picture displays a pellet of BL21 (DE3) with pCons-eforRed in UV-light. The right picture is a pellet of BL21 (DE3) in white light.


Further characterization was performed in order to demonstrate the absorbance and fluorescence of eforRed. BL21 (DE3) containing pCons-eforRed were spread on a petri dish containing 25 µg/ml chloramphenicol and was photographed in white light and on an UV-table emitting 302 nm (Figure 2). The results were the same as above, in white light (Figure 2, right) the cultures had a burgundy color and on the UV-table the eforRed expressing bacteria exhibited a pink glowing colour (Figure 2, left).


T--Linkoping Sweden--eforred-agar-photo.png
Figure 2. Colonies in the same host as previously is presented in white light (right side) and in 302 nm UV-light (left side).

























To test the oxygen dependency of the protein production of eforRed in BL21 (DE3), the bacteria containing pCons-eforRed was grown O.N. to 2 OD600 and diluted to 0.49 OD600 with LB-miller. The bacteria was placed in a 96-well plate in replicates of 4 with 200 µL in each well. The 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. A spectrometry experiment was conducted measuring the fluorescence (excitation 589, emission 609) in 37 °C for 24 hours and the experiment (Figure 3) showed that the access to oxygen effects the folding of eforRed and that 4 holes gave the highest yield.


T--Linkoping Sweden--efffforedd.png T--Linkoping Sweden--eforredvsmngphoto.jpeg


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.

T--Linkoping Sweden--eforred SDS-page.png

















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