Difference between revisions of "Part:BBa K2609006"
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     <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 idea of imroving the part.</p>
 
     <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 idea of imroving the part.</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>
−
     <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/a/ab/T--IISc-Bangalore--imcherry_sds.png" width=100% style="border: 1px solid black;">
+
     <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>
−
     <figcaption>SDS PAGE with the cell lysate, Supernatant after binding with the Ni-NTA beads, Wash , and Elution. The top band is the non-truncated protein and the thee bottom band is the truncated protein.</figcaption>
+
<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; ">
−
     </figure>  
+
         <img src="https://static.igem.org/mediawiki/parts/2/24/T--IISc-Bangalore--linkTest.svg-imCherLys.png" width=100% style="border: 1px solid black;">
−
    <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 cells 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 25 kDa and 32 kDa. The heavier band is the non-truncated protein and the lighter one is the truncated protein. </p>
+
     <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 thus 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 purification would have the non-truncated protein.</p>
+
 
−
    <figure <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;">
+
     <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>
−
         <img src="https://static.igem.org/mediawiki/parts/9/9f/T--IISc-Bangalore--imcherry_excitation_emission.png" width=100% style="border: 1px solid black;">
+
<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;">
−
        <figcaption>Tht excitation and emission spectra of imCherry after normalizing it with WT BL21 (DE3) lysate.<hr>
+
         <img src="https://static.igem.org/mediawiki/parts/6/65/T--IISc-Bangalore--imCherySDSpuri.png" width=100% style="border: 1px solid black;">
−
        Note: The negative fluoroscense in the graph are due to the normalization procedure.
+
    <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>
−
        </figcaption>
+
     </figure> </center>
−
     </figure>    
+
     
 
     <h3>Fluoroscence</h3>
 
     <h3>Fluoroscence</h3>
 +
     
 
     <h4 style="font-weight:900">Excitation Spectrum</h4>
 
     <h4 style="font-weight:900">Excitation Spectrum</h4>
−
     <p>The excitaion spectrum of the purified sample(elution) was obtained at a fixed emission wavelength of 610 nm. The excitation maxima was obtained at 576 nm.</p>
+
     <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>
 
     <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 607 nm</p>
+
     <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>
−
     <h3>Quantification of Truncation</h3>
+
 
 +
<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;">
 +
        <figcaption>Tht excitation and emission spectra of imCherry 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 fluoroscence.
 +
        </figcaption>
 +
    </figure>
 +
<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>
 
     <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.</li>
−
         <li>By combining the fluorescense 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 fluoresence. The fluorescence of each of the above fractions were divided into fluorescnce due to truncated and non-truncated protein based on their coressponding 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>
+
         <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>

Revision as of 09:16, 13 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 fluroscent protein mcherry (BBa_J18932). 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%.(http://2018.igem.org/Team:IISc-Bangalore/Improve)

Usage

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 idea of imroving the part.

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 fluoroscence.
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.
  • 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

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]