Difference between revisions of "Part:BBa K864402"

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===Usage and Biology===
  
 
===Contribution===
 
===Contribution===
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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.
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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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<li> </html>the [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034]Ribosome binding site<html></li>
 
<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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<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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<p> To verify eforRed's absorbance and emission, the construct was expressed in <i>E. coli </i> BL21 (DE3). The bacteria containing the constitutive promotor (pCons) was compared to a negative control (<b>Figure 1</b>). Thereafter the eforRed expressing bacteria was centrifuged, resulting in a pellet with a burgundy color. (<b>Figure 1</b>, top-right corner). To demonstrate the fluorescence of eforRed, the pellet was placed on a UV-table emitting a wavelength of 302 nm, (<b>Figure 1</b>, down-right corner), which exhibited a pink glowing colour. 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.</p>
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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>
 
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<div class="figurtext"style=font-size:80%;><i><b>Figure 1.</b> The picture to the left depict a <i>E. coli</i> BL21 (DE3) culture expressing pCons-eforRed (left tube) versus a negative control (right tube) with <i>E. coli</i> BL21 (DE3) containing no construct after 48 hours incubation in 37°C. The top right picture displays centrifuged cultures of <i>E. coli</i> BL21 (DE3) with pCons-eforRed in UV-light. The right picture is a culture of <I>E. coli</I> BL21 (DE3) expressing eforRed which has been incubated for 48 hours in 37 degrees Celsius and centrifuged at 12 000g 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).</i></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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Further characterization was performed in order to demonstrate the absorbance and fluorescence of <i>E. coli</i> BL21 (DE3) containing pCons-eforRed. An eforRed culture was spread on an LB-agar plate containing 25 µg/ml chloramphenicol. The agar culture were photographed in visual light and on a UV-table emitting 302 nm (figure 2). The results were the same as above, in visual light (<b>Figure 2</b>, right) the cultures had a burgundy color and on the UV-table the bacteria exhibited a pink glowing colour (<b>Figure 2</b>, left).
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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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</html>[[Image:T--Linkoping_Sweden--eforred-agar-photo.png|700px|thumb|left|<div class="figurtext"style=font-size:80%;><i><b>Figure 2.</b> <i> E. coli</i> 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|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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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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<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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<div class="liucontent" style=font-size:80%;><i><b>Figure 3.</b></i> To the left is a of platereading <i>E. coli</i> 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 <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.</i></div>
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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>
 
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>
 
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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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><div class="figurtext"style=font-size:80%;><b><i>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.</i>
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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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===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