Difference between revisions of "Part:BBa K1073022:Experience"

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===Applications of BBa_K1073022===
 
===Applications of BBa_K1073022===
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When eforRed is expressed, the colonies on agar plates as well as liquid culture exhibit a bright pink color.
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The iGEM Team Braunschweig 2013 used this device as a reporter to distinguish two different strains when cultivated in a mixed culture. <br> When eforRed is expressed, the colonies on agar plates as well as the liquid culture exhibit a bright pink color.
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The iGEM Team Braunschweig 2013 used this device to distinguish two different strains when cultivation in a mixed culture.
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===Part Sequence Quality===
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'''The Tech Museum, May 2014''': This part's real sequence may end with two stop codons, TAATAA. This is not listed in [https://parts.igem.org/Part:BBa_K592012 the original source part's sequence] or in this part's sequence, but [https://parts.igem.org/cgi/sequencing/one_blast.cgi?update_database=1&id=19622 sequencing from the registry] indicates it may be present. Beware.
  
 
===User Reviews===
 
===User Reviews===
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<I>iGEM Team Braunschweig 2013</I>
 
<I>iGEM Team Braunschweig 2013</I>
 
|width='60%' valign='top'|
 
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When expressed the chromoprotein eforRed exhibits a distinct pink color. In order to avoid absorption of the chomoproteins during measurement with the photometer the absorption and emission spectra were measured and an optimal wavelength for spectral optical density measurement was determined according to these spectra.  
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When expressed, the chromoprotein eforRed exhibits a distinct pink color. In order to avoid absorption by the chomoprotein during OD measurement of a bacterial culture, the absorption and emission spectra were measured. An optimal wavelength for spectral optical density measurement was determined to be at 520nm.  
  
 
[[File:BSAbsorption-eforRed.jpg |400px]]
 
[[File:BSAbsorption-eforRed.jpg |400px]]
  
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'''iGEM Team Braunschweig 2013:''' Absorptionspectrum of chromoprotein eforRed in the wavelength range 400-800 nm.
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'''iGEM Team Braunschweig 2013:''' Absorption spectrum of chromoprotein eforRed in the wavelength range 400-800 nm.
 
   
 
   
 
'''Absorption maximum of eforRed:''' 534 nm
 
'''Absorption maximum of eforRed:''' 534 nm
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[[File:BS2013Emission eforRed.jpg |400px]]
 
[[File:BS2013Emission eforRed.jpg |400px]]
  
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'''iGEM Team Braunschweig 2013:''' Emission spectrum of chromoprotein eforRed. This chromoprotein does show significant emission and can be detected by its fluorescence as well as by naked eye.  
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'''iGEM Team Braunschweig 2013:''' Emission spectrum of chromoprotein eforRed. The chromoprotein does show significant emission and can be detected by its fluorescence.  
  
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'''Emission maximum of eforRed:''' 605,4 nm  
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'''Emission maximum of eforRed:''' 605 nm <br>
  
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According to these results the optical density for growth curves of cells expressing this protein was measured at 520 nm with respect to the chromoproteins amilGFP (<partinfo>BBa_K1073024</partinfo>)  and aeBlue (<partinfo>BBa_K1073020</partinfo>) to avoid absorption by the proteins. Comparing all absorption spectra they all show an absorption minimum at 520 nm.
 
  
 
[[File:Fluorescence.JPG |200px]]
 
[[File:Fluorescence.JPG |200px]]
  
 
'''iGEM Team Braunschweig 2013:''' Supernatant after cell disruption containing chromoproteins of eforRed and amilGFP (<partinfo>BBa_K1073024</partinfo>). Both chromoproteins  fluoresce when excited by UV-light.
 
'''iGEM Team Braunschweig 2013:''' Supernatant after cell disruption containing chromoproteins of eforRed and amilGFP (<partinfo>BBa_K1073024</partinfo>). Both chromoproteins  fluoresce when excited by UV-light.
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|};
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<partinfo>BBa_K1073022 AddReview 5</partinfo>
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<I>iGEM Team Pasteur Paris 2019</I>
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To further characterize this biobrick, we decided to focus on the fluorescence expression of eforRed in ''E. coli''.
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'''Fluorescence microscopy'''
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This experiment was performed using an RFP filter. Samples were analyzed after 30 hours of incubation and compared to a non-transformed ''E. coli'' strain.
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[[File:T--Pasteur Paris--Fluorescence_microscopy_eforRed_1.png |500px]]
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We can clearly see that BBa_K1073022 allows the expression of red fluorescence in ''E. coli'' after several hours of incubation.
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However, when we merged brightfield and mRFP images, we observed that the fluorescence is not expressed by all of the bacteria.
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[[File:T--Pasteur Paris--Fluorescence_microscopy_eforRed_2.png |700px]]
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'''Flow cytometry'''
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In addition of fluorescence microscopy, we performed flow cytometry in order to test the efficiency of transformation and the stability of the expression of eforRed over time.
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[[File:T--Pasteur Paris--Flow_cytometry.jpg |800px]]
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Less than 50% of bacteria expressed the red fluorescence after 2 hours of incubation, and this percentage decreases over time.
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This low percentage of expression could be explained by a poor transformation efficiency. When the selective pressure was removed, the bacteria continued to lose expression of the chromoprotein as wild type bacteria out-competed those with the plasmid.
  
 
|};
 
|};
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K1073022 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K1073022 EndReviews</partinfo>

Latest revision as of 00:07, 22 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_K1073022

The iGEM Team Braunschweig 2013 used this device as a reporter to distinguish two different strains when cultivated in a mixed culture.
When eforRed is expressed, the colonies on agar plates as well as the liquid culture exhibit a bright pink color.


Part Sequence Quality

The Tech Museum, May 2014: This part's real sequence may end with two stop codons, TAATAA. This is not listed in the original source part's sequence or in this part's sequence, but sequencing from the registry indicates it may be present. Beware.

User Reviews

UNIQbc699e6fde5b6a11-partinfo-00000000-QINU

•••••

iGEM Team Braunschweig 2013

When expressed, the chromoprotein eforRed exhibits a distinct pink color. In order to avoid absorption by the chomoprotein during OD measurement of a bacterial culture, the absorption and emission spectra were measured. An optimal wavelength for spectral optical density measurement was determined to be at 520nm.

BSAbsorption-eforRed.jpg

iGEM Team Braunschweig 2013: Absorption spectrum of chromoprotein eforRed in the wavelength range 400-800 nm.

Absorption maximum of eforRed: 534 nm

BS2013Emission eforRed.jpg

iGEM Team Braunschweig 2013: Emission spectrum of chromoprotein eforRed. The chromoprotein does show significant emission and can be detected by its fluorescence.

Emission maximum of eforRed: 605 nm


Fluorescence.JPG

iGEM Team Braunschweig 2013: Supernatant after cell disruption containing chromoproteins of eforRed and amilGFP (BBa_K1073024). Both chromoproteins fluoresce when excited by UV-light.

;
•••••

iGEM Team Pasteur Paris 2019

To further characterize this biobrick, we decided to focus on the fluorescence expression of eforRed in E. coli.

Fluorescence microscopy

This experiment was performed using an RFP filter. Samples were analyzed after 30 hours of incubation and compared to a non-transformed E. coli strain.


T--Pasteur Paris--Fluorescence microscopy eforRed 1.png


We can clearly see that BBa_K1073022 allows the expression of red fluorescence in E. coli after several hours of incubation.

However, when we merged brightfield and mRFP images, we observed that the fluorescence is not expressed by all of the bacteria.


T--Pasteur Paris--Fluorescence microscopy eforRed 2.png


Flow cytometry

In addition of fluorescence microscopy, we performed flow cytometry in order to test the efficiency of transformation and the stability of the expression of eforRed over time.


T--Pasteur Paris--Flow cytometry.jpg


Less than 50% of bacteria expressed the red fluorescence after 2 hours of incubation, and this percentage decreases over time.

This low percentage of expression could be explained by a poor transformation efficiency. When the selective pressure was removed, the bacteria continued to lose expression of the chromoprotein as wild type bacteria out-competed those with the plasmid.

;

UNIQbc699e6fde5b6a11-partinfo-00000004-QINU