Difference between revisions of "Part:BBa K2609016"

 
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This part generates imCherry (an improved version of BBa_J18932 with reduced truncation) when transformed into a T7 expression strain like Bl21 (DE3).
 
This part generates imCherry (an improved version of BBa_J18932 with reduced truncation) when transformed into a T7 expression strain like Bl21 (DE3).
  
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===Usage and Biology===
 
  
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===Usage and Biology===
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<html>
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    <h2>Biology</h2>
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    <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>
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    <h2>Usage</h2>
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    <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>
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    <h2>Characterization</h2>
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    <h3>Expression with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609016>BBa_K2609016</a></h3>
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    <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>
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    <h3> Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609016>BBa_K2609016</a> </h3>
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    <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>
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    <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;">
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        <img src="https://static.igem.org/mediawiki/parts/9/9f/T--IISc-Bangalore--imcherry_excitation_emission.png" width=100% style="border: 1px solid black;">
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        <figcaption>Tht excitation and emission spectra of imCherry after normalizing it with WT BL21 (DE3) lysate.<hr>
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        Note: The negative fluoroscense in the graph are due to the normalization procedure.
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        </figcaption>
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    </figure>   
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    <h3>Fluoroscence</h3>
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    <h4 style="font-weight:900">Excitation Spectrum</h4>
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    <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>
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    <h4 style="font-weight: 900">Emission Spectrum</h4>
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    <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>
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    <h3>Quantification of Truncation</h3>
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    <p> The truncation of imCherry was determined by through two orthogonal methods:</p>
 +
    <ul>
 +
        <li>By analaysing the intensity of the truncated and non-truncated protein bands in the SDS PAGE.</li>
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        <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>
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    </ul>
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    <h4>Truncation Data</h4>
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        text-align: center;
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    }
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</html>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 22:31, 11 October 2018


6xHis-imCherry under T7 expression system

This part generates imCherry (an improved version of BBa_J18932 with reduced truncation) when transformed into a T7 expression strain like Bl21 (DE3).


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 improvement of the part.

Characterization

Expression with BBa_K2609016

The protein was expressed under T7 promoter in E.coliBL21(DE3) with 6x-Histag at the N-terminal. The culture was induced at 37°C for three hours with IPTG concentration of 500μM. 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

Purification using Ni-NTA with BBa_K2609016

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.

Tht excitation and emission spectra of imCherry after normalizing it with WT BL21 (DE3) lysate.
Note: The negative fluoroscense in the graph are due to the normalization procedure.

Fluoroscence

Excitation Spectrum

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.

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

Quantification of Truncation

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

  • By analaysing the intensity of the truncated and non-truncated protein bands in the SDS PAGE.
  • 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.

Truncation Data

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 768
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]