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| | ===Usage and Biology=== | | ===Usage and Biology=== |
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| − | <h2>Using this as a T7 expression backbone</h2>
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| − | <h3>Expressing any protein of interest</h3>
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| − | <p>This T7 expression backbone can be used to express any protein if its coding sequence (with a start codon) is inserted using the HindIII and NheI sites. These sites can be added to the coding sequence using PCR with primers having 5'-overhangs.</p>
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| − | <h3>Fusing a protein domain at the N-terminus of an existing protein</h3>
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| − | <p>By inserting the coding sequence of a protein domain (including the start codon) using the HindIII and AgeI sites into the T7 expression backbone (which already contains a protein coding sequence), an N-terminal fusion can be performed.</p>
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| − | <h2>Expressing a protein of interest</h2>
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| − | <p>No system is better for protein expression than E. coli BL21 (DE3) as its lon and ompT protease deficiency yields a huge amount of protein. BL21 (DE3) is a lysogenic strain that has the T7 RNA polymerase gene integrated into its genome under the lac operon; adding IPTG induces expression of T7 RNA polymerase, which recognizes the T7 promoter sequence. Any gene inserted downstream of the T7 promoter can thus be expressed.</p>
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| − | <p>Transform this part into BL21 (DE3) and induce protein overexpression with IPTG after the culture reaches early-log to mid-log phase (optimize this by inducing at different times).</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_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°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>
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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; ">
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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: 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/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>576 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>607 nm</b></p>
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| − | <center>
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| − | <figure style="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;">
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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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| − | <hr>
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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 after SDS PAGE.</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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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
