Difference between revisions of "Team:NAIT Edmonton/Desc"

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<link href='http://fonts.googleapis.com/css?family=Source+Sans+Pro' rel='stylesheet' type='text/css'>
 
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<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script>
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<script type="text/javascript" src="https://2015.igem.org/Team:NAIT_Edmonton/jquery.flexslider-min?action=raw&amp;ctype=text/javascript"></script>
  
 
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$(document).ready(function() {
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                 <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Team">team</a>
 
                 <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Team">team</a>
 
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                 <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Project">project</a>
 
                 <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Project">project</a>
 
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                 <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Human">human practices</a>
 
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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>
 
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Revision as of 18:08, 10 August 2015

Team NAIT 2015

The Project

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, including the previously colour-coded molecular weight markers. 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 molecular weight markers with the novel capacity to provide users colour-coded bands post-staining. Our technology will also pave the way for new types of colorimetric assays that will provide less expensive and more accessible reporter and tagging systems.


Background
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 separate and identify specific proteins of interest. Among these techniques, SDS-PAGE plays an essential role due to its high sensitivity, low sample volume requirement, and high popularity. Negatively charged proteins migrate towards the positive electrode according to their size and charge. Smaller proteins migrate further in a given amount of time. As proteins are separated in this manner, users load molecular weight standards to estimate the size (in kDa) of the proteins present in their sample. Once the proteins of a single sample have been isolated and are embedded in the polyacrylamide (PA) gel matrix, staining procedures are used to visualize them.



Organic dyes, such as Coomassie blue, can be used for this purpose; nevertheless, their low sensitivity and a detection range that goes from 1 to 50 ng can be a challenge for detecting low abundance proteins (Jin, Huang, Yoo, & Choi, 2006). A higher sensitivity can be achieved by fluorescent staining techniques (from 0.1 to 10 ng.); however, UV instruments are necessary in order to read the data (Jin et al., 2006). The most sensitive method up to date is radiolabeling, but the requirement of hazardous isotopes and their complex management makes it a complicated procedure (Jin et al., 2006). Silver staining is a method that offers great sensitivity and an easy to handle protocol, thus making it one of the most commonly used staining methods.


The Problem

Difficulties with silver staining arise when the molecular weight markers are re- colored golden-brown in the staining process. Markers offer evenly distributed proteins 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 subset of these markers has color-coded standard proteins to facilitate the identification of each band. Post-silver staining, the users lose the ability to use the color code as a reference.


Our Goal

Our goal is to develop a marker that, when interacting with the reagents used in the staining protocol, will develop colour bands in specific positions so as to help in the identification of the protein(s) of interest post-staining. In order to do so, investigation of how specific amino acids react with silver staining reagents is underway by our team. This will have as an outcome the creation of novel proteins that contain an excess of a particular amino acid and/or chemical modifications that will generate a specific colour after treating it with silver staining reagents. To obtain such proteins, the introduction of novel nucleotide sequences into a plasmid would be done first by in vitro transcription translation and later by transforming E. coli cells with expression vectors.

Our Solution

Design novel protein sequences that will stain in colour.

Why Does this Matter?

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.