Difference between revisions of "Part:BBa K864402:Experience"

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<h2> 2019 iGEM team Linkoping Sweden </h2>
 
<h2> 2019 iGEM team Linkoping Sweden </h2>
  
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2019 iGEM team Linkoping Sweden validated this part.<br><br>
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2019 iGEM team Linkoping Sweden validated this part.<br>
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Summary:
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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 <i>E. coli </i> BL21 (DE3) cells. The molecular weight of eforRed was also examined with an SDS-PAGE.<br>
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<b style="font-size:120%;">Fluorescence and absorbance</b><br>
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To verify eforReds absorbance and emission, the construct was heat shocked into <i>Escherichia coli</i>, 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.</p>
  
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[[Image:T--Linkoping_Sweden--pcons-eforred.jpeg|460px]]
 
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[[Image:T--Linkoping_Sweden--eforrudtsgfytdpl.jpeg|460px]]
 
 
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<i><b>Figure 1.</b></i> <b>To the left</b> This biobrick after 48 hours in 37 degrees Celsius (both pictures). To the left is cultured E. coli BL21 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. <b>To the right</b> is colonies in the same host as before illuminated in 302 nm UV-light, showing eforReds ability to also emit strong fluorescence.
 
  
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</html>[[Image:T--Linkoping_Sweden--pcons-eforred.jpeg|460px]]<html>
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</html>[[Image:T--Linkoping_Sweden--eforred-pellet-photo.png|400px]]<html>
 
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[[Image:T--Linkoping_Sweden--eforredoxygen.png|460px]]
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<div class="figurtext" style=font-size:90%;><b>Figure 1.</b> 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.</div>
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[[Image:T--Linkoping_Sweden--eforredvsmngphoto.jpeg|460px]]
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<i><b>Figure 2.</b></i> <b>To the left</b> 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. <b>To the right</b> 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.
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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).
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<p>[[Image:T--Linkoping_Sweden--eforred_SDS-page.png|100px|left]] bla bla bla</p>
 
  
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</html>[[Image:T--Linkoping_Sweden--eforred-agar-photo.png|700px|left|thumb|<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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<b style="font-size:120%;">Oxygen dependency</b>
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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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</html>[[Image:T--Linkoping_Sweden--efffforedd.png|460px]]<html>
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</html>[[Image:T--Linkoping_Sweden--eforredvsmngphoto.jpeg|460px]]<html>
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<b>Figure 3.</b> To the left is a spectrometry experiment of BL21´s (DE3) protein production of eforRed with varying access to oxygen. The y-axis depicts the relative fluorescence intensity and the x-axis represents the time up to 24 hours. The plate with eforRed can be seen on the right pictures left side and the one on the right side is <i>E.coli</i> 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.
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<p><b style="font-size:120%;">Molecular weight</b><br>
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A study of the molecular weight of pCons eforRed expressed in <i>E.coli</i> BL21 (DE3)  was done by sonicating the cells and  performing a SDS-PAGE electrophoresis on the lysate (<b>Figure 4.</b>). Biorads "Precision Plus Protein Dual Color Standards" was used as the protein ladder in the electrophoresis.<br><br>
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</html>[[Image:T--Linkoping_Sweden--eforred_SDS-page.png|100px|left|]]<html>
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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<div class="figurtext"style=font-size:80%;>
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<b>Figure 4.</b> SDS-page of sonicated <I>E.coli</I> 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.
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===User Reviews===
 
===User Reviews===

Latest revision as of 17:02, 20 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K864402

2019 iGEM team Linkoping Sweden

2019 iGEM team Linkoping Sweden validated this part.

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 an SDS-PAGE.

Fluorescence and absorbance
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).

Figure 2. Colonies in the same host as previously is presented in white light (right side) and in 302 nm UV-light (left side).
























Oxygen dependency 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 spectrometry experiment of BL21´s (DE3) protein production of eforRed with varying access to oxygen. The y-axis depicts the relative fluorescence intensity and the x-axis represents the time up to 24 hours. The plate with eforRed can be seen on the right pictures left side and the one on the right side 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.


Molecular weight
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.

User Reviews

UNIQa34f874a3ff30e53-partinfo-00000007-QINU UNIQa34f874a3ff30e53-partinfo-00000008-QINU