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− | {{NAIT_Edmonton/CSS2}} | + | {{NAIT_Edmonton/CSS6}} |
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− | <style type="text/css">
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− | <li><a href="https://igem.org/Team.cgi">official team profile</a> | + | <li><a href="https://igem.org/Team.cgi?id=1787">official team profile</a> |
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| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Desc">description</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Desc">description</a></li> |
− | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Protocols">experiment and protcols</a></li> | + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Protocols">experiment and protocols</a></li> |
| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Results">parts and results</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Results">parts and results</a></li> |
| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Modeling">modeling</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Modeling">modeling</a></li> |
− | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Software">software</a></li>
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− | <div class="header_right">
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− | <ul>
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| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Human">human practices</a> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Human">human practices</a> |
| <ul> | | <ul> |
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| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Practices">policy and practices</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Practices">policy and practices</a></li> |
| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Collaborations">collaborations</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Collaborations">collaborations</a></li> |
− | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Entrepreunership">entrepreunership</a></li> | + | |
| </ul> | | </ul> |
| </li> | | </li> |
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| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Notebook">notebook</a> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Notebook">notebook</a> |
| <ul> | | <ul> |
| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Safety">lab safety</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Safety">lab safety</a></li> |
− | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Measurement">measurement</a></li> | + | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Achievements">achievements</a></li> |
| <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Logbook">log book</a></li> | | <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Logbook">log book</a></li> |
| </ul> | | </ul> |
| </li> | | </li> |
− | </ul>
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− | </div>
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| </div> | | </div> |
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| + | </div> |
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| + | <div id="wrap"> |
| + | <div class="wrapper"> |
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| + | <style type="text/css"> |
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| + | .top_slogan {text-align:center; font-family: 'Source Sans Pro', sans-serif; color:#0D4D8C; font-size:30px; padding: 40px 0px 40px 0px; font-style:strong; line-height:40px;} |
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| + | width: 580px; |
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| + | font-size: 1.0em; |
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| + | overflow:hidden; |
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| + | padding:20px 80px 20px 80px; |
| + | display:none; |
| + | font-size:18px; |
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| + | |
| + | .protocol {background-color:#6087c2; width:390px;margin:0 auto; -webkit-border-radius: 5px; |
| + | -moz-border-radius:5px; -ms-border-radius:5px; -0-border-radius:5px; |
| + | border-radius: 5px;padding:15px;} |
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| + | border-radius: 5px;padding:15px;} |
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| + | </style> |
| + | |
| + | <center><div class="top_slogan">Development and Characterization of Protein Motifs to Generate Colours upon Interaction with Silver Staining Reagents</div></center> |
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− | <div id="wrap">
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− | <div class="main_content">
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− | <center><div class="top_slogan">The Project</div></center> | + | <h2><b>Abstract: </b>SDS-PAGE is a very popular technique used to separate proteins based on their size. Embedded proteins, invisible to the naked eye, are then visualized by staining. Among the various staining techniques, silver staining is easy to perform and highly sensitive. However, the outcome is a series of monochromatic protein bands. Previously, we observed that some proteins inherently produce different hues post-staining. We hypothesized that specific amino acid configurations yield coloured bands after reacting with silver staining reagents. To test our hypothesis, we created numerous amino acid motifs to elucidate the sequences that would generate specific colours following silver staining. Our findings will let us generate a molecular weight marker with the innate capacity of providing users colour-coded bands post-staining without the use of impregnating dyes. Our technology will also pave the way for new types of colorimetric assays using synthetic proteins</h2> <br> |
| | | |
− | <h1>Background</h1></a> | + | <div class="accordion"> |
− |
| + | <div class="accordion-section"> |
− | <p>The structural and functional study of the proteins expressed by a genome is
| + | <a class="accordion-section-title" href="#accordion-1" style="background-color:#f96040">Background</a> |
| + | <div id="accordion-1" class="accordion-section-content"> |
| + | The structural and functional study of the proteins expressed by a genome is |
| | | |
| called proteomics. This relatively novel science uses different methodologies in order to | | called proteomics. This relatively novel science uses different methodologies in order to |
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| <br> | | <br> |
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− | <center><img src="http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png" width="750px"></center> | + | <center><img src="https://static.igem.org/mediawiki/2015/b/bb/NAIT_SDSPAGE.png" width="750px"></center> |
| + | <p style="font-size:10px">Source: http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png</p> |
| | | |
| <br> | | <br> |
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− | <p>Organic dyes, such as Coomassie blue, can be used for this purpose; | + | <p style="font-size:15;">Organic dyes, such as Coomassie blue, can be used for this purpose; |
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| nevertheless, their low sensitivity and a detection range that goes from 1 to 50 ng can | | nevertheless, their low sensitivity and a detection range that goes from 1 to 50 ng can |
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| <br> | | <br> |
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− | <center><img src="http://www.bio-rad.com/webroot/web/images/lsr/products/electrophoresis/product_overlay_content/global/lsr_biosafe_coomasie_gel.jpg"></center> | + | <center><img src="https://static.igem.org/mediawiki/2015/b/b0/Lsr_biosafe_coomasie_gel.jpg"></center> |
| + | <p><font size="1px">[1] Bio-rad.com, 'Coomassie Stains', 2015. [Online]. Available: http://www.bio-rad.com/webroot/web/images/lsr/products/electrophoresis/product_overlay_content/global/lsr_biosafe_coomasie_gel.jpg. [Accessed: 14- Jun- 2015].</font></p> |
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− | <br><br> | + | </div><!--end .accordion-section-content--> |
| + | </div><!--end .accordion-section--> |
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− | <h1>The Problem </h1></a> | + | <div class="accordion-section"> |
− | | + | <a class="accordion-section-title" href="#accordion-2" style="background-color:#FBB252">The Problem</a> |
− | <p>Difficulties with silver staining arise when the molecular weight markers are re- | + | <div id="accordion-2" class="accordion-section-content"> |
− | | + | <p>However, there are limitations to using the silver staining technique. The primary issue with the technique is that the users lose the ability to use the color coded molecular weight marker as a reference post-staining. To combat this issue, researchers poke holes into the polyacrylamide gel so that they can retain their molecular weight ladder reference points. However, by doing this we ruin the integrity of the gel making the staining process much more likely to damage or destroy the fragile gel. </p> |
− | colored golden-brown in the staining process. Markers offer evenly distributed proteins
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− | | + | |
− | that show bands of equal intensity and known size. Researchers can compare these
| + | |
− | | + | |
− | bands with their sample and identify the protein they are looking for based on its size. A
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− | | + | |
− | subset of these markers has color-coded standard proteins to facilitate the identification
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− | | + | |
− | of each band. Post-silver staining, the users lose the ability to use the color code as a
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− | | + | |
− | reference.</p> | + | |
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| <br> | | <br> |
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− | <center><img src="http://labs.mmg.pitt.edu/gjoerup/silver_stain.jpg" width="750px"></center> | + | <center><img src="https://static.igem.org/mediawiki/2015/a/a9/NAIT_SilverStain.jpeg" width="750px"> |
| + | <p style="font-size:10px">Source:http://labs.mmg.pitt.edu/gjoerup/silver_stain.jpg</p> |
| + | </center><br> |
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− | <br><br> | + | <p>Researchers who have used the technique in the past have also expressed concerns on the time it takes to develop the gel and stain; the possibility of “over-staining” the gel, and the generation of false positives due to the sensitivity of the technique. (An example would be the solutions reacting with particles in the air to provide an unneeded background on the gel) </p> |
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− | <h1>Our Goal</h1></a> | + | </div><!--end .accordion-section-content--> |
| + | </div><!--end .accordion-section--> |
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− | <p>Our goal is to develop a marker that, when interacting with the reagents used in | + | <div class="accordion-section"> |
| + | <a class="accordion-section-title" href="#accordion-3" style="background-color: #f7e133">Our Goal and Solution</a> |
| + | <div id="accordion-3" class="accordion-section-content"> |
| + | <p>Our goal is to develop a marker that, when interacting with the reagents used in the staining protocol, will stain in colour and in specific positions. This technology will aid in the identification of the protein(s) of interest post-staining. In order to do so, we will investigate how specific amino acids react with silver staining reagents. After determining specific motifs, we aim to create novel proteins that contain an excess of that motif, particular amino acid and/or chemical modifications that will generate a specific color after treating it with silver staining reagents. To obtain such proteins, we will introduce novel nucleotide sequences into a plasmid by in vitro transcription translation. <i>E. coli</i> cells with expression vectors will then be transformed with the new plasmids.</p> <br> |
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− | the staining protocol, will develop colour bands in specific positions so as to help in the | + | <p>By testing different techniques to develop and stain the gels we hope to see if the expressed concerns are still an issue, namely time taken to develop the gels and stain them effectively. To investigate this we will use BioRad’s recommended protocol, our protocol developed in-house, and a technique documented in <i>Electrophoresis.</i></p> |
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− | identification of the protein(s) of interest post-staining. In order to do so, investigation of
| + | </div><!--end .accordion-section-content--> |
| + | </div><!--end .accordion-section--> |
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− | how specific amino acids react with silver staining reagents is underway by our team.
| + | <div class="accordion-section"> |
| + | <a class="accordion-section-title" href="#accordion-5" style="background-color:#afcf64">Why Does this Matter?</a> |
| + | <div id="accordion-5" class="accordion-section-content"> |
| + | <p>With the current technique used today, researchers poke holes into the PA gel so that they can retain their molecular weight ladder reference points. Not only does this take time to do, but it also ruins the integrity of the gel making the staining process much more likely to damage the fragile gel.</p> |
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− | This will have as an outcome the creation of novel proteins that contain an excess of a
| + | </div><!--end .accordion-section-content--> |
| + | </div><!--end .accordion-section--> |
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− | particular amino acid and/or chemical modifications that will generate a specific colour
| + | </div><!--end .accordion--> |
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− | after treating it with silver staining reagents. To obtain such proteins, the introduction of
| |
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− | novel nucleotide sequences into a plasmid would be done first by in vitro transcription
| + | |
| + | <div class="push"></div> |
| + | </div> |
| + | |
| + | </div> |
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− | translation and later by transforming E. coli cells with expression vectors.</p>
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| <li><a href="http://www.facebook.com/pages/IGEM-2015-NAIT-Edmonton/884025281659292"><img src="https://static.igem.org/mediawiki/2015/a/a3/NAIT_Icon_facebook.png" alt="" title="" /></a></li> | | <li><a href="http://www.facebook.com/pages/IGEM-2015-NAIT-Edmonton/884025281659292"><img src="https://static.igem.org/mediawiki/2015/a/a3/NAIT_Icon_facebook.png" alt="" title="" /></a></li> |
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− | ul.footer_menu li{ float:left; padding:0 15px 0 0;}
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− | ul.footer_menu li a{ background:url(https://static.igem.org/mediawiki/2015/1/17/NAIT_Bullet.png) no-repeat left; padding:0 0 0 12px;}
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− |
| |
− |
| |
− | </style>
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| | | |
| </body> | | </body> |
| | | |
− |
| |
− | </body>
| |
| </html> | | </html> |