Difference between revisions of "Part:BBa K864402"
 
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===Usage and Biology===
  
 
===Contribution===
 
===Contribution===
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<br>
 
<br>
 
Summary:  
 
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 chromophore protein expression in E.coli BL21(DE3) cells and then measured the absorbance of the cells with a plate reader.
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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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Documentation:
 
Documentation:
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<html>
 
<html>
  
 
The expression system used  contained the following parts:
 
The expression system used  contained the following parts:
 
<ul>
 
<ul>
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<li> </html>the [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] ribosome binding site<html></li>
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<li> </html>the [https://parts.igem.org/Part:BBa_J23110 BBa_J23110]Constitutive promotor<html></li>
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<li> </html>the [https://parts.igem.org/Part:BBa_J23110 BBa_J23110] Constitutive promotor<html></li>
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<li> </html>the [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034]Ribosome binding site<html></li>
 
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<p>
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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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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.
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</html>[[Image:T--Linkoping_Sweden--pcons-eforred.jpeg|460px]]<html>
 
</html>[[Image:T--Linkoping_Sweden--pcons-eforred.jpeg|460px]]<html>
 
</html>[[Image:T--Linkoping_Sweden--eforred-pellet-photo.png|400px]]<html>
 
</html>[[Image:T--Linkoping_Sweden--eforred-pellet-photo.png|400px]]<html>
 
<br><br>
 
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<div class="figurtext"style=font-size:80%;><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 (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. <b>In the bottom right </b> 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 <b>top right </b> was put on a UV table (302 nm) which resulted in a pink glowing pellet.</div>
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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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<br><br>
 
<br><br>
  
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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.
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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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<br><br>
 
<br><br>
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<div>
 
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</html>[[Image:T--Linkoping_Sweden--eforred-agar-photo.png|left|thumb|700px|<b><i>Figure 2.</i></b> 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.]]<html>
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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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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
 
<div>
 
<div>
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<p>
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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>
 
</div>
 
</div>
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<div>
 
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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.
 
  
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<div>
 
</html>[[Image:T--Linkoping_Sweden--efffforedd.png|460px]]<html>
 
</html>[[Image:T--Linkoping_Sweden--efffforedd.png|460px]]<html>
 
</html>[[Image:T--Linkoping_Sweden--eforredvsmngphoto.jpeg|460px]]<html>
 
</html>[[Image:T--Linkoping_Sweden--eforredvsmngphoto.jpeg|460px]]<html>
 
</div>
 
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<i><b>Figure 3.</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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<div style=font-size:90%;>
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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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</div>
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<br><br>
 
<br><br>
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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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</p>
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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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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>
 
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</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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===Usage and Biology===
 
  
 
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Latest revision as of 16:51, 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 an SDS-PAGE.
Documentation:

The expression system used contained the following parts:

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.



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