Difference between revisions of "Part:BBa K2609006"
 
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An improved alternative to mCherry (BBa_J18932) that reduces the truncation by 50% improving usage in fusion proteins.
 
An improved alternative to mCherry (BBa_J18932) that reduces the truncation by 50% improving usage in fusion proteins.
−
 
−
 
−
 
−
 
 
===Usage and Biology===
 
===Usage and Biology===
 
<html>
 
<html>
 
     <h2>Biology</h2>
 
     <h2>Biology</h2>
−
     <p> imCherry is an improved version of the fluroscent protein mcherry (<a href=https://parts.igem.org/Part:BBa_J18932>BBa_J18932</a>. mcherry has truncation at its N-terminal due to a strong RBS sequence in front of the the ninth amino acid, Met. The new part, imCherry is made by modifying this RBS sequence such its translational efficiency is reduced, which reduces truncation. The new part is shown to have a reduction in truncation by about 50%.<a href=http://2018.igem.org/Team:IISc-Bangalore/Improve>http://2018.igem.org/Team:IISc-Bangalore/Improve</a></p>
+
     <p> imCherry is an improved version of the fluorescent protein mCherry (<a href="https://parts.igem.org/Part:BBa_J18932">BBa_J18932</a>) which is a widely used marker for protein studies. A fusion at the N-term of mCherry however is not a viable method for quantification because of the prominent truncation suffered by the protein near this terminal. This is caused by the presence of a strong RBS sequence upstream of the the ninth amino acid, a methionine encoded by a start codon that acts as a site of initiation. The new part, imCherry is made by modifying this internal RBS sequence such that its translational efficiency is reduced, which thereby reduces truncation. The new part is shown to have truncation reduced by about 50%.(<a href=http://2018.igem.org/Team:IISc-Bangalore/Improve>http://2018.igem.org/Team:IISc-Bangalore/Improve</a>)</p>
 +
 
 
     <h2>Usage</h2>
 
     <h2>Usage</h2>
−
     <p> The idea of Imcherry came into the picture when the 2018 IISc-Bangalore iGem team decided to check the efficieny of their N-terminal signal peptide PelB using mCherry. They found out that mcherry gets truncated by about 50%, which led to the improvement of the part.</p>
+
     <p> The mCherry part,<a href="https://parts.igem.org/Part:BBa_J18932">BBa_J18932</a>, was to be used by the 2018 IISc-Bangalore iGEM team for characterization of a N-term signal peptide when they realised that an estimate obtained from truncation prone mCherry would not be accurate. imCherry was created to answer this problem. The sequence at the internal RBS was modified in-silico, and the truncation characterized experimentally using a combination of PAGE and Fluorescent quantification. (See <a href=http://2018.igem.org/Team:IISc-Bangalore/Improve>http://2018.igem.org/Team:IISc-Bangalore/Improve</a>).</p>
−
    <h2>Characterization</h2>
+
 
 +
    <h2>Characterization</h2>
 +
     
 +
 
 
     <h3>Expression with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609016>BBa_K2609016</a></h3>
 
     <h3>Expression with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609016>BBa_K2609016</a></h3>
−
     <p> The protein was expressed under T7 promoter in <i>E.coli</i>BL21(DE3) with 6x-Histag at the N-terminal. The culture was induced at 37&deg;C for three hours with IPTG concentration of 500&mu;M. The cell were then lysed to obatin the protein The size of the complete protein with 6x-Histag is about 26kDa. We observed two bands in the induced sample between </p>
+
   
 +
     <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: 20%; 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/2/24/T--IISc-Bangalore--linkTest.svg-imCherLys.png" width=100% style="border: 1px solid black;">
 +
    <figcaption>SDS PAGE with the cell lysate. 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_K2609016>BBa_K2609016</a> </h3>
 
     <h3> Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609016>BBa_K2609016</a> </h3>
−
     <p>The cell lysate obatined was purified using Ni-NTA beads which only binds to the proteins with 6x-Histag, which is absent in the truncated protein. So the supernatant after binding would have the truncated protein and the elution after the purification would have the non-truncated protein.</p>
+
 
 +
     <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: 30%; 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/6/65/T--IISc-Bangalore--imCherySDSpuri.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>
 
     <h3>Fluoroscence</h3>
−
     <h4>Excitation Spectrum</h4>
+
     
−
     <figure style="float: right; width: 50%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em;">
+
     <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>576 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>607 nm</b></p>
 +
 
 +
<figure style="float:left; width: 45%; 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/9/9f/T--IISc-Bangalore--imcherry_excitation_emission.png" width=100% style="border: 1px solid black;">
 
         <img src="https://static.igem.org/mediawiki/parts/9/9f/T--IISc-Bangalore--imcherry_excitation_emission.png" width=100% style="border: 1px solid black;">
−
         <figcaption><a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a> expressed in BL21 (DE3) at 25°C for 16 hrs. 1mL of the culture was resuspended in 100uL PBS and mixed with 100uL of 2xSDS Sample Buffer followed by boiling. 20uL of the final mix was loaded onto a SDS-PAGE gel.</figcaption>
+
         <figcaption>Tht excitation and emission spectra of imCherry after normalizing it with WT BL21 (DE3) lysate.<hr>
−
     </figure>    
+
        Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluorescence.
−
    <p>The excitaion spectrum of the purified sample(elution) was obtained at a fixed emission wavelength of 610 nm.</p>
+
        </figcaption>
−
     <h3>Quantification of Truncation</h3>
+
    </figure>
−
     <p> The truncation of imCherry was determined by through two orthogonal methods:</p>
+
<figure style="float:right; width: 45%; 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/7/7a/T--IISc-Bangalore--imCherryplates.png" width=100% style="border: 1px solid black;">
 +
        <figcaption>mCherry and imCherry colonies on media lacking IPTG. imCherry seems to have a higher basal fluorescence in the absence of an inducer. This might be due to a increase in over translation rate from the proper initiation site after the removal of the internal RBS.
 +
     
 +
        </figcaption>
 +
     </figure>
 +
<hr>
 +
     <h3 style="clear:both;">Quantification of Truncation</h3>
 +
     <p> The truncation of imCherry was determined by through two different methods:</p>
 
     <ul>
 
     <ul>
−
         <li>By analaysing the intensity of the truncated and non-truncated protein bands in the SDS PAGE.</li>
+
         <li>By analysing the intensity of the truncated and non-truncated protein bands after SDS PAGE of the crude lysate.</li>
−
         <li>By combining the fluorescense and gel intensity data of the Ni-NTA purification products(supernatant after binding, wash and elution).This is done assuming that truncated and non-truncated protein has the same fluoresence. The fluorescence of each of the above samples were divided into fluorescnce due to truncated and non-truncated based on their coressponding band intensities. The sum of fluorescence values of truncated and non-truncated were then used as a measure of their concentration to determine truncation.</li>
+
         <li>By combining the fluorescence and gel intensity data of the Ni-NTA purification products (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 samples 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>
 
     </ul>
−
    <h4>Truncation Data</h4>
+
  <h4>Truncation Data</h4>
 +
 
 +
<p><u>From gel intensity (rough estimation)</u>:</p>
 +
 
 +
<table style="width: 45%; border-collapse: collapse; float:left;">
 +
    <tr style="background-color: #9e9e9e">
 +
        <td style="border-left: none; border-bottom: none"></td>
 +
        <td colspan="2">% of protein (imCherry)</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> 19.9 </td>
 +
        <td> 80.1 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 2</td>
 +
        <td> 34.2 </td>
 +
        <td> 65.8 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 3</td>
 +
        <td> 24 </td>
 +
        <td> 76 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 4</td>
 +
        <td> 21.7 </td>
 +
        <td> 78.3 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 5</td>
 +
        <td> 20 </td>
 +
        <td> 80 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Average</td>
 +
        <td> 23.96 </td>
 +
        <td> 76.04 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Std.dev</td>
 +
        <td> 5.33 </td>
 +
        <td> 5.33 </td>
 +
    </tr>
 +
</table>
 +
 
 +
<figure style="width: 34%; 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/1/11/T--IISc-Bangalore--imcherry_trun_gel.png" width=100% style="border: 1px solid black;">
 +
        <figcaption>The scatter plot showing the replicates, average and standard deviation for imCherry and mCherry with gel data. From the statistical significance analysis of the two means, we got a p-value of P=0.0062 with (**).
 +
        </figcaption>
 +
    </figure>
 +
<br><br><br><br>
 +
<p style="clear:left; display: inline-block; margin-top: 150px; font-size:15px;"><b> From this, imcherry is estimated to have a truncation of 23.96 % &plusmn; 5.33 %</b></p>
 +
 
 +
 
 +
 +
<p style="clear:right;"><u>From the calculations combining gel intensity and fluorescense</u>:</p>
 +
<table style="width: 45%; border-collapse: collapse; float:left;">
 +
    <tr style="background-color: #9e9e9e">
 +
        <td style="border-left: none; border-bottom: none"></td>
 +
        <td colspan="2">% of protein (imCherry)</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> 16.35 </td>
 +
        <td> 83.65 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 2</td>
 +
        <td> 22.82 </td>
 +
        <td> 77.18 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 3</td>
 +
        <td> 20.53 </td>
 +
        <td> 79.47 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 4</td>
 +
        <td> 21.81 </td>
 +
        <td> 78.19 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Replicate 5</td>
 +
        <td> 20.74 </td>
 +
        <td> 79.26 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Average</td>
 +
        <td> 20.45 </td>
 +
        <td> 79.55 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Std.dev</td>
 +
        <td> 2.21 </td>
 +
        <td> 2.21 </td>
 +
    </tr>
 +
</table>
 +
 
 +
<figure style="width: 34%; 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/c/cb/T--IISc-Bangalore--imcherry_trun_gel_flu.png" width=100% style="border: 1px solid black;">
 +
        <figcaption>The scatter plot showing the replicates, average and standard deviation for imCherry and mCherry with gel+fluorescense data. From the statistical significance analysis of the two means, we got a p-value of P<0.0001 with (***).
 +
        </figcaption>
 +
    </figure>
 +
<br><br><br><br>
 +
<p style="clear:left; display: inline-block; margin-top: 150px; margin-bottom: 40px; font-size:15px;"><b> From this, imcherry is estimated to have a truncation of 20.45 % &plusmn; 2.21 %</b></p>
 +
<h4>Truncation reduction</h4>
 +
    <p> From the fine measurements combining gel intensity data and fluorescense data, mCherry is observed to have truncation of 38.82 % &plusmn; 2.52 % (From <a href=https://parts.igem.org/Part:BBa_K2319009>BBa_K2319009</a>). The improved version of mcherry, imCherry has truncation of 20.45 % &plusmn; 2.21 %. Therefore, the percentage reduction of truncation in imCherry from mCherry is about 47% ( statistical significance analysis gave p<0.0001) within the error limits. </p>
 +
<hr>
 +
<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>
 +
 
 
<style>
 
<style>
 
     table, td, td{
 
     table, td, td{
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</style>
 
</style>
 
</html>
 
</html>
 +
    <hr>
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 21:28, 17 October 2018


imCherry (improved mCherry)

An improved alternative to mCherry (BBa_J18932) that reduces the truncation by 50% improving usage in fusion proteins.

Usage and Biology

Biology

imCherry is an improved version of the fluorescent protein mCherry (BBa_J18932) which is a widely used marker for protein studies. A fusion at the N-term of mCherry however is not a viable method for quantification because of the prominent truncation suffered by the protein near this terminal. This is caused by the presence of a strong RBS sequence upstream of the the ninth amino acid, a methionine encoded by a start codon that acts as a site of initiation. The new part, imCherry is made by modifying this internal RBS sequence such that its translational efficiency is reduced, which thereby reduces truncation. The new part is shown to have truncation reduced by about 50%.(http://2018.igem.org/Team:IISc-Bangalore/Improve)

Usage

The mCherry part,BBa_J18932, was to be used by the 2018 IISc-Bangalore iGEM team for characterization of a N-term signal peptide when they realised that an estimate obtained from truncation prone mCherry would not be accurate. imCherry was created to answer this problem. The sequence at the internal RBS was modified in-silico, and the truncation characterized experimentally using a combination of PAGE and Fluorescent quantification. (See http://2018.igem.org/Team:IISc-Bangalore/Improve).

Characterization

Expression with BBa_K2609016

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. The top band is the non-truncated protein and the the bottom band is the truncated protein.

Purification using Ni-NTA with BBa_K2609016

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 576 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 607 nm

Tht excitation and emission spectra of imCherry 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.
mCherry and imCherry colonies on media lacking IPTG. imCherry seems to have a higher basal fluorescence in the absence of an inducer. This might be due to a increase in over translation rate from the proper initiation site after the removal of the internal RBS.

Quantification of Truncation

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

  • By analysing the intensity of the truncated and non-truncated protein bands after SDS PAGE of the crude lysate.
  • By combining the fluorescence and gel intensity data of the Ni-NTA purification products (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 samples 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 (imCherry)
Truncated Non-Truncated
Replicate 1 19.9 80.1
Replicate 2 34.2 65.8
Replicate 3 24 76
Replicate 4 21.7 78.3
Replicate 5 20 80
Average 23.96 76.04
Std.dev 5.33 5.33
The scatter plot showing the replicates, average and standard deviation for imCherry and mCherry with gel data. From the statistical significance analysis of the two means, we got a p-value of P=0.0062 with (**).




From this, imcherry is estimated to have a truncation of 23.96 % ± 5.33 %

From the calculations combining gel intensity and fluorescense:

% of protein (imCherry)
Truncated Non-Truncated
Replicate 1 16.35 83.65
Replicate 2 22.82 77.18
Replicate 3 20.53 79.47
Replicate 4 21.81 78.19
Replicate 5 20.74 79.26
Average 20.45 79.55
Std.dev 2.21 2.21
The scatter plot showing the replicates, average and standard deviation for imCherry and mCherry with gel+fluorescense data. From the statistical significance analysis of the two means, we got a p-value of P<0.0001 with (***).




From this, imcherry is estimated to have a truncation of 20.45 % ± 2.21 %

Truncation reduction

From the fine measurements combining gel intensity data and fluorescense data, mCherry is observed to have truncation of 38.82 % ± 2.52 % (From BBa_K2319009). The improved version of mcherry, imCherry has truncation of 20.45 % ± 2.21 %. Therefore, the percentage reduction of truncation in imCherry from mCherry is about 47% ( statistical significance analysis gave p<0.0001) within the error limits.

For more information see Team:IISc-Bangalore/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]