Difference between revisions of "Part:BBa J18932"
(Undo revision 391762 by Adwaith (talk))
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* Best maturation for expression at 20 or 25 C (rather than 37)
 
* Best maturation for expression at 20 or 25 C (rather than 37)
  
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===Usage and Biology===
 
===Usage and Biology===
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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_K2319009>BBa_K2319009</a></h3>
 
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    <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>
 
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<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; ">
 
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        <img src="https://static.igem.org/mediawiki/parts/8/85/T--IISc-Bangalore--mcherry-expression.png" width=100% style="border: 1px solid black;">
 
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    <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>
 
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    </figure></center>
 
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    <h3> Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a> </h3>
 
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    <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>
 
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<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;">
 
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        <img src="https://static.igem.org/mediawiki/parts/8/8f/T--IISc-Bangalore--mcherry-Truncation.png" width=100% style="border: 1px solid black;">
 
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    <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>
 
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    </figure> </center>
 
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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 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>
 
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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 <b>605 nm</b></p>
 
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<center>
 
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<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;">
 
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        <img src="https://static.igem.org/mediawiki/parts/1/1e/T--IISc-Bangalore--mcherry_excitation_emission.png" width=100% style="border: 1px solid black;">
 
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        <figcaption>Tht excitation and emission spectra of mCherry after normalizing it with WT BL21 (DE3) lysate.<hr>
 
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        Note: The kinks in the graph are an artifact of the normalization procedure to eliminate source fluoroscence.
 
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        </figcaption>
 
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    </figure>
 
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    </center>
 
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    <h3 style="clear:both;">Quantification of Truncation</h3>
 
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    <p> The truncation of mCherry was determined by through two different methods:</p>
 
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    <ul>
 
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        <li>By analysing the intensity of the truncated and non-truncated protein bands from the SDS PAGE of the crdue lysate.(rough estimation)</li>
 
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        <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>
 
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    </ul>
 
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<h4>Truncation Data</h4>
 
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<p><u>From gel intesity (rough esimation)</u>:</p>
 
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<table style="width: 70%; border-collapse: collapse; float:left;">
 
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    <tr style="background-color: #9e9e9e">
 
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        <td style="border-left: none; border-bottom: none"></td>
 
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        <td colspan="2">% of protein</td>
 
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    </tr>
 
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    <tr style="background-color: #9e9e9e">
 
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        <td style="border-left: none; border-top: none"></td>
 
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        <td>Truncated</td>
 
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        <td>Non-Truncated</td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 1</td>
 
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        <td> 30.25 </td>
 
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        <td> 69.75 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 2</td>
 
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        <td> 48.6 </td>
 
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        <td> 51.4 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 3</td>
 
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        <td> 35.8 </td>
 
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        <td> 64.2 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 4</td>
 
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        <td> 34.2 </td>
 
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        <td> 65.8 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 5</td>
 
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        <td> 49.8 </td>
 
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        <td> 50.2 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Average</td>
 
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        <td> 39.73 </td>
 
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        <td> 60.27 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Std.dev</td>
 
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        <td> 7.95 </td>
 
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        <td> 7.95 </td>
 
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    </tr>
 
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</table>
 
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<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;">
 
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        <img src="https://static.igem.org/mediawiki/parts/d/d4/T--IISc-Bangalore--mcherry_trun_gel.png" width=100% style="border: 1px solid black;">
 
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        <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with gel data.
 
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        </figcaption>
 
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    </figure>
 
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<br><br><br><br>
 
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<p style="clear:left; display: inline-block; margin-top: 150px;"><b> From this, mcherry is estimated to have a truncation of 39.73 % +/- 7.95 %</b></p>
 
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<p style="clear:right;"><u>From the calculations combining gel intensity and fluorescense</u>:</p>
 
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<table style="width: 70%; border-collapse: collapse; float:left;">
 
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    <tr style="background-color: #9e9e9e">
 
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        <td style="border-left: none; border-bottom: none"></td>
 
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        <td colspan="2">% of protein</td>
 
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    </tr>
 
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    <tr style="background-color: #9e9e9e">
 
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        <td style="border-left: none; border-top: none"></td>
 
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        <td>Truncated</td>
 
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        <td>Non-Truncated</td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 1</td>
 
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        <td> 34.66 </td>
 
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        <td> 65.34 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 2</td>
 
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        <td> 38.91 </td>
 
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        <td> 61.09 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 3</td>
 
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        <td> 42.53 </td>
 
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        <td> 57.47 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 4</td>
 
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        <td> 39.51 </td>
 
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        <td> 60.49 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Replicate 5</td>
 
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        <td> 38.47 </td>
 
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        <td> 61.53 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Average</td>
 
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        <td> 38.82 </td>
 
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        <td> 61.18 </td>
 
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    </tr>
 
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    <tr>
 
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        <td>Std.dev</td>
 
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        <td> 2.52 </td>
 
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        <td> 2.53 </td>
 
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    </tr>
 
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</table>
 
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<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;">
 
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        <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;">
 
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        <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescnse data.
 
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        </figcaption>
 
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    </figure>
 
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<br><br><br><br>
 
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<p style="clear:left; display: inline-block; margin-top: 150px; margin-bottom: 150px;"><b> From this, mcherry is estimated to have a truncation of 38.82 % +/- 2.52 %</b></p>
 
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<hr>
 
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<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>
 
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</html>
 
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Revision as of 22:09, 16 October 2018

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

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]