Difference between revisions of "Part:BBa J18932"
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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) | ||
− | + | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
+ | <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°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. </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 fluoroscence. | ||
+ | </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 crdue 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 intesity (rough esimation)</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;"><b> From this, mcherry is estimated to have a truncation of 39.73 % +/- 7.95 %</b></p> | ||
+ | |||
+ | |||
+ | |||
+ | <p style="clear:right;"><u>From the calculations combining gel intensity and fluorescense</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+Fluorescnse data. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <br><br><br><br> | ||
+ | <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> | ||
+ | <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> | ||
+ | table, td, td{ | ||
+ | border: 1px solid black; | ||
+ | |||
+ | } | ||
+ | td{ | ||
+ | text-align: center; | ||
+ | } | ||
+ | </style> | ||
+ | |||
+ | </html> | ||
+ | <hr> | ||
<!-- --> | <!-- --> |
Revision as of 22:08, 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
Characterization
Expression with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a>
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.
<img src="" 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>
Purification using Ni-NTA with <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K2319009>BBa_K2319009</a>
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.
<img src="" 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>
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
<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="" width=100% style="border: 1px solid black;"><figcaption>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 fluoroscence. </figcaption> </figure>
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 crdue 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 intesity (rough esimation):
% 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 |
<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="" 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>
From this, mcherry is estimated to have a truncation of 39.73 % +/- 7.95 %
From the calculations combining gel intensity and fluorescense:
% 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 |
<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="" width=100% style="border: 1px solid black;"> <figcaption>The scatter plot showing the replicates, average and standard deviation for mCherry with Gel+Fluorescnse data. </figcaption> </figure>
From this, mcherry is estimated to have a truncation of 38.82 % +/- 2.52 %
<style>
table, td, td{ border: 1px solid black; } td{ text-align: center; }
</style>
</html>
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]