Difference between revisions of "Part:BBa J18932"
 
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* Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli
 
* Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli
−
* Contains a hidden translation start site in the N-terminal -> up to 50% of protein produced in E.coli will be truncated
+
* Contains a hidden translation start site in the N-terminal -> up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)
−
* SDS treatment and boiling before PAGE hydrolyzes the chromophore at F//MYG splitting the protein in half
+
* SDS treatment and boiling before PAGE hydrolyzes the chromophore at F//MYG splitting the protein in half (discussed in Gross et al. (2000) The structure of the chromophore within DsRed protein from coral.)
 
* Best maturation for expression at 20 or 25 C (rather than 37)
 
* Best maturation for expression at 20 or 25 C (rather than 37)
  
−
<!-- Add more about the biology of this part here
+
<!-- -->
 
===Usage and Biology===
 
===Usage and Biology===
 +
<html>
 +
    <h2>Characterization</h2>
 +
     
 +
 +
    <h3>Expression with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a></h3>
 +
   
 +
    <p> The protein was expressed under T7 promoter in <i>E.coli</i> BL21(DE3) with 6x-His tag at the N-terminal. The culture was induced at 37&deg;C for three hours with a final IPTG concentration of 500&mu;M. The cells were then lysed to obtain the protein. The size of the complete protein with 6x-Histag is about 26kDa. We observed two bands in the induced sample between 25 kDa and 32 kDa. The heavier band is the non-truncated protein and the lighter one is its truncated counterpart. </p>
 +
<center><figure style=" width: 35%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em; ">
 +
        <img src="https://static.igem.org/mediawiki/parts/8/85/T--IISc-Bangalore--mcherry-expression.png" width=100% style="border: 1px solid black;">
 +
    <figcaption>SDS PAGE with the cell lysate for WT uninduced, WT induced, K2319009 uninduced, K2319009 induced. The top band is the non-truncated protein and the the bottom band is the truncated protein.</figcaption>
 +
    </figure></center>
 +
     
 +
 +
    <h3> Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a> </h3>
 +
 +
    <p>The cell lysate thus obtained was purified using Ni-NTA beads which only bind to proteins with a 6x-His tag, which is absent in the truncated protein. Ideally, the supernatant after binding should have the truncated protein and the elution after purification should have the non-truncated protein. This however is not true because the binding of 6xHis to Ni-NTA is not perfect.</p>
 +
<center><figure style=" width: 40%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em; clear:right;">
 +
        <img src="https://static.igem.org/mediawiki/parts/8/8f/T--IISc-Bangalore--mcherry-Truncation.png" width=100% style="border: 1px solid black;">
 +
    <figcaption>SDS PAGE of fractions from Ni-NTA purification. The top band is the non-truncated protein and the bottom band is the protein truncated at the internal start codon (see arrowheads).</figcaption>
 +
    </figure> </center>
 +
     
 +
    <h3>Fluoroscence</h3>
 +
     
 +
    <h4 style="font-weight:900">Excitation Spectrum</h4>
 +
    <p>The excitation spectrum of the purified sample (elution) was obtained at a fixed emission wavelength of 610 nm. The excitation maxima was obtained at <b>567 nm</b>.</p>
 +
    <h4 style="font-weight: 900">Emission Spectrum</h4>
 +
    <p>The emission spectrum of the purified sample (elution) was obtained at a fixed excitation wavelength of 587 nm. The emission maxima was obtained at <b>605 nm</b></p>
 +
<center>
 +
<figure style="width: 50%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em; clear:right;">
 +
        <img src="https://static.igem.org/mediawiki/parts/1/1e/T--IISc-Bangalore--mcherry_excitation_emission.png" width=100% style="border: 1px solid black;">
 +
        <figcaption>Tht excitation and emission spectra of mCherry after normalizing it with WT BL21 (DE3) lysate.<hr>
 +
        Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluorescence.
 +
        </figcaption>
 +
    </figure>
 +
    </center>
 +
 +
    <h3 style="clear:both;">Quantification of Truncation</h3>
 +
    <p> The truncation of mCherry was determined by through two different methods:</p>
 +
    <ul>
 +
        <li>By analysing the intensity of the truncated and non-truncated protein bands from the SDS PAGE of the crude lysate.(rough estimation)</li>
 +
        <li>By combining the fluorescence and gel intensity data of the Ni-NTA purification fractions (supernatant after binding, wash and elution).This is done assuming that truncated and non-truncated protein has the same fluorescence. The fluorescence of each of the above fractions was divided into fluorescence due to truncated and non-truncated protein based on their corresponding band intensities. The sum of fluorescence values of truncated and non-truncated protein were then used as a measure of their concentration to determine truncation.</li>
 +
    </ul>
 +
<h4>Truncation Data</h4>
 +
 +
<p><u>From gel intensity (rough estimation)</u>:</p>
 +
 +
<table style="width: 70%; border-collapse: collapse; float:left;">
 +
    <tr style="background-color: #9e9e9e">
 +
        <td style="border-left: none; border-bottom: none"></td>
 +
        <td colspan="2">% of protein</td>
 +
    </tr>
 +
    <tr style="background-color: #9e9e9e">
 +
        <td style="border-left: none; border-top: none"></td>
 +
        <td>Truncated</td>
 +
        <td>Non-Truncated</td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 1</td>
 +
        <td> 30.25 </td>
 +
        <td> 69.75 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 2</td>
 +
        <td> 48.6 </td>
 +
        <td> 51.4 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 3</td>
 +
        <td> 35.8 </td>
 +
        <td> 64.2 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 4</td>
 +
        <td> 34.2 </td>
 +
        <td> 65.8 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 5</td>
 +
        <td> 49.8 </td>
 +
        <td> 50.2 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Average</td>
 +
        <td> 39.73 </td>
 +
        <td> 60.27 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Std.dev</td>
 +
        <td> 7.95 </td>
 +
        <td> 7.95 </td>
 +
    </tr>
 +
</table>
 +
 +
<figure style="width: 20%; float:right; max-height: 300px text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; padding: 0.5em;">
 +
        <img src="https://static.igem.org/mediawiki/parts/d/d4/T--IISc-Bangalore--mcherry_trun_gel.png" width=100% style="border: 1px solid black;">
 +
        <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.
 +
        </figcaption>
 +
    </figure>
 +
<br><br><br><br>
 +
<p style="clear:left; display: inline-block; margin-top: 150px; font-size:15px;"><b> From this, mcherry is estimated to have a truncation of 39.73 % &plusmn; 7.95 %</b></p>
 +
 +
 +
 +
<p style="clear:right;"><u>From the calculations combining gel intensity and fluorescence</u>:</p>
 +
<table style="width: 70%; border-collapse: collapse; float:left;">
 +
    <tr style="background-color: #9e9e9e">
 +
        <td style="border-left: none; border-bottom: none"></td>
 +
        <td colspan="2">% of protein</td>
 +
    </tr>
 +
    <tr style="background-color: #9e9e9e">
 +
        <td style="border-left: none; border-top: none"></td>
 +
        <td>Truncated</td>
 +
        <td>Non-Truncated</td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 1</td>
 +
        <td> 34.66 </td>
 +
        <td> 65.34 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 2</td>
 +
        <td> 38.91 </td>
 +
        <td> 61.09 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 3</td>
 +
        <td> 42.53 </td>
 +
        <td> 57.47 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 4</td>
 +
        <td> 39.51 </td>
 +
        <td> 60.49 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 5</td>
 +
        <td> 38.47 </td>
 +
        <td> 61.53 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Average</td>
 +
        <td> 38.82 </td>
 +
        <td> 61.18 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Std.dev</td>
 +
        <td> 2.52 </td>
 +
        <td> 2.53 </td>
 +
    </tr>
 +
</table>
 +
 +
<figure style="width: 20%; float:right; max-height: 300px text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; padding: 0.5em;">
 +
        <img src="https://static.igem.org/mediawiki/parts/f/f4/T--IISc-Bangalore--mCherry_flu_gel_trunc.png" width=100% style="border: 1px solid black;">
 +
        <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescense data.
 +
        </figcaption>
 +
    </figure>
 +
<br><br><br><br>
 +
<p style="clear:left; display: inline-block; margin-top: 150px; margin-bottom: 150px; font-size:15px;"><b> From this, mcherry is estimated to have a truncation of 38.82 % &plusmn; 2.52 %</b></p>
 +
<div style="display: block; width:35%;";>For more information see <a href="http://2018.igem.org/Team:IISc-Bangalore/Improve">Team:IISc-Bangalore/Improve</a></div>
 +
<hr>
 +
<h3>Improvement by IISc-Bangalore 2018</h3>
 +
The sequence was modified to reduce the strength of the internal RBS and made into <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006">BBa_K2609006</a>.
 +
<br><br>
 +
<h3>Improvement by IISc-Bangalore 2019</h3>
 +
The sequence was modified to reduce the truncation by replacement of the internal start codon with Isoleucine and made into <a href="https://parts.igem.org/Part:BBa_K3165013">BBa_K3165013</a>.
 +
<br><br>
 +
<h3>Improvement by NYCU-Taipei 2022</h3>
 +
The sequence was modified by adding a tag and made into <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K4461013">BBa_K4461013</a>
 +
<div style="display: block; width:35%;";>For more information see <a href="https://2022.igem.wiki/nycu-taipei/improve">Team:NYCU-Taipei/Improve</a></div>
 +
<br>
 +
 +
<style>
 +
    table, td, td{
 +
        border: 1px solid black;
 +
       
 +
    }
 +
    td{
 +
        text-align: center;
 +
    }
 +
</style>
 +
   
 +
</html>
  
 
<!-- -->
 
<!-- -->
Line 28: Line 210:
 
<partinfo>BBa_J18932 parameters</partinfo>
 
<partinfo>BBa_J18932 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
 +
==Contribution: UniGE-Geneva==
 +
'''Group:''' UniGE-Geneva
 +
<br>'''Authors:''' Grégory Segala and Bryan Bourrat
 +
<br>'''Summary:'''In the list of Parts assigned to our team, we decided to characterize the fluorescent protein : mCherry RFP (BBa-J18932). Our goal was to observe the evolution of the intensity of fluorescence emitted by this protein, after induction by IPTG (Isopropyl -D-1-thiogalactopyranoside), varying two parameters: temperature and pH.
 +
<br>'''Documentation:'''
 +
<br>
 +
 +
[[Image:T--UniGE-Geneva--unige-igem-characterization-2.004.png|frame|none|'''Fig 1''':Procedure details of the experiment.]]
 +
[[Image:T--UniGE-Geneva--unige-igem-characterization-2.005.png|frame|none|'''Fig 2''':Variation of Fluorescence Intensity depending on Temperature.]]
 +
[[Image:T--UniGE-Geneva--unige-igem-characterization-2.006.png |frame|none|'''Fig 3''':Variation of Fluorescence Intensity depending on pH.]]
 +
 +
<br>'''Conclusion:''' We can say that when the temperature increase, the fluorescence of mCherry decrease quite sharply. But, It is also observed that there is no variation in the intensity of fluorescence,  depending on whether you are in an acidic, neutral or alkaline pH environment. Thus, mCherry seems less stable than the other fluorescent protein we characterized (sfGFP : BBa-I746916).

Latest revision as of 12:27, 11 October 2022

mCherry RFP

Red fluorescent protein derived from DsRed.

Advantages:

  • fast folding and maturation
  • bright and photo-stable

Purity issues (update):

  • Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli
  • Contains a hidden translation start site in the N-terminal -> up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)
  • SDS treatment and boiling before PAGE hydrolyzes the chromophore at F//MYG splitting the protein in half (discussed in Gross et al. (2000) The structure of the chromophore within DsRed protein from coral.)
  • Best maturation for expression at 20 or 25 C (rather than 37)

Usage and Biology

Characterization

Expression with BBa_K2319009

The protein was expressed under T7 promoter in E.coli BL21(DE3) with 6x-His tag at the N-terminal. The culture was induced at 37°C for three hours with a final IPTG concentration of 500μM. The cells were then lysed to obtain the protein. The size of the complete protein with 6x-Histag is about 26kDa. We observed two bands in the induced sample between 25 kDa and 32 kDa. The heavier band is the non-truncated protein and the lighter one is its truncated counterpart.

SDS PAGE with the cell lysate for WT uninduced, WT induced, K2319009 uninduced, K2319009 induced. The top band is the non-truncated protein and the the bottom band is the truncated protein.

Purification using Ni-NTA with BBa_K2319009

The cell lysate thus obtained was purified using Ni-NTA beads which only bind to proteins with a 6x-His tag, which is absent in the truncated protein. Ideally, the supernatant after binding should have the truncated protein and the elution after purification should have the non-truncated protein. This however is not true because the binding of 6xHis to Ni-NTA is not perfect.

SDS PAGE of fractions from Ni-NTA purification. The top band is the non-truncated protein and the bottom band is the protein truncated at the internal start codon (see arrowheads).

Fluoroscence

Excitation Spectrum

The excitation spectrum of the purified sample (elution) was obtained at a fixed emission wavelength of 610 nm. The excitation maxima was obtained at 567 nm.

Emission Spectrum

The emission spectrum of the purified sample (elution) was obtained at a fixed excitation wavelength of 587 nm. The emission maxima was obtained at 605 nm

Tht excitation and emission spectra of mCherry after normalizing it with WT BL21 (DE3) lysate.
Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluorescence.

Quantification of Truncation

The truncation of mCherry was determined by through two different methods:

  • By analysing the intensity of the truncated and non-truncated protein bands from the SDS PAGE of the crude lysate.(rough estimation)
  • By combining the fluorescence and gel intensity data of the Ni-NTA purification fractions (supernatant after binding, wash and elution).This is done assuming that truncated and non-truncated protein has the same fluorescence. The fluorescence of each of the above fractions was divided into fluorescence due to truncated and non-truncated protein based on their corresponding band intensities. The sum of fluorescence values of truncated and non-truncated protein were then used as a measure of their concentration to determine truncation.

Truncation Data

From gel intensity (rough estimation):

% of protein
Truncated Non-Truncated
Replicate 1 30.25 69.75
Replicate 2 48.6 51.4
Replicate 3 35.8 64.2
Replicate 4 34.2 65.8
Replicate 5 49.8 50.2
Average 39.73 60.27
Std.dev 7.95 7.95
The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.




From this, mcherry is estimated to have a truncation of 39.73 % ± 7.95 %

From the calculations combining gel intensity and fluorescence:

% of protein
Truncated Non-Truncated
Replicate 1 34.66 65.34
Replicate 2 38.91 61.09
Replicate 3 42.53 57.47
Replicate 4 39.51 60.49
Replicate 5 38.47 61.53
Average 38.82 61.18
Std.dev 2.52 2.53
The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescense data.




From this, mcherry is estimated to have a truncation of 38.82 % ± 2.52 %

For more information see Team:IISc-Bangalore/Improve

Improvement by IISc-Bangalore 2018

The sequence was modified to reduce the strength of the internal RBS and made into BBa_K2609006.

Improvement by IISc-Bangalore 2019

The sequence was modified to reduce the truncation by replacement of the internal start codon with Isoleucine and made into BBa_K3165013.

Improvement by NYCU-Taipei 2022

The sequence was modified by adding a tag and made into BBa_K4461013
For more information see Team:NYCU-Taipei/Improve

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Contribution: UniGE-Geneva

Group: UniGE-Geneva
Authors: Grégory Segala and Bryan Bourrat
Summary:In the list of Parts assigned to our team, we decided to characterize the fluorescent protein : mCherry RFP (BBa-J18932). Our goal was to observe the evolution of the intensity of fluorescence emitted by this protein, after induction by IPTG (Isopropyl -D-1-thiogalactopyranoside), varying two parameters: temperature and pH.
Documentation:

Fig 1:Procedure details of the experiment.
Fig 2:Variation of Fluorescence Intensity depending on Temperature.
Fig 3:Variation of Fluorescence Intensity depending on pH.


Conclusion: We can say that when the temperature increase, the fluorescence of mCherry decrease quite sharply. But, It is also observed that there is no variation in the intensity of fluorescence, depending on whether you are in an acidic, neutral or alkaline pH environment. Thus, mCherry seems less stable than the other fluorescent protein we characterized (sfGFP : BBa-I746916).