Difference between revisions of "Team:CU-Boulder/Model"

 
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<div class = "mainhead"><h><i> &#8226; InterLab &#8226;</i></h></div>
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<div class = "mainhead"><h><i> &#8226; Modeling &#8226;</i></h></div>
  
 
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This year our team decided to participate in the 2017 Interlab study, a multi-lab study in standardization for instrument measurements. More information can be found at the<a href="https://2017.igem.org/Competition/InterLab_Study"> 2017 iGEM Website</a>. Our interlab notebook can be found at <a href="https://2017.igem.org/Team:CU-Boulder/InterLabNoteBook"> here</a>. Our instrument was a BioTek Synergy 2 plate reader. </p>
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In order to increase our efficiency, while saving time and resources, the team undertook various techniques in molecular modeling in order to discern the best places to perform point mutations. These three separate mechanisms are discussed in detail below. The 14 mutations that we eventually decided on can be seen here in red. Five of these mutations have been carried over from last year, along with nine new mutations added with this year's project. </p>
 
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<i>Many thanks to the Detweiler and Junge labs here at CU for providing some materials and equipment access without which we would not have been able to perform this protocol, and to our mentor Dr. Brian DeDecker for training on the plate reader</i>.
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&#8226; References  &#8226;  
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&#8226; Length & Fit : Intramolecular &#8226;  
  
 
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<h> OD600 Reference Point </h>
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One of the first approaches that we took in modeling was the knowledge if the approximate difference in length of our AzoPhe residue between its cis and trans conformation. We can see the individual monomers of our hexameric protein in here.</p>
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<h>Well Layout </h>
 
  
 
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If we found individual residues that were positioned between individual monomers such that the distance between the two were greater than <b>6 Angstroms</b> (the overall length of our residue in its cis conformation, and less than <b>13 Angstroms</b> (the overall length of our residue in the trans conformation), then we can reason that our hexamers will be unable to pack once being activated at this point, leaving us to believe that this would be a good position for a point mutation.</p>
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&#8226; LUDOX & Standardization &#8226;
 
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For these we required no deviations from the standard protocol. A small amount of pipetting error seems to have been present in the fluorescein measurements. We hope to minimize this in the cell protocol by using reverse pipetting. We believe our standard curves were linear enough to not require repeating, however.</p>
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As an example, we can see how this works for one of our point mutations. We see that when we zoom in, that there is a measured distance of <b> 7.1 Angstroms </b>  between our residue of interest and the other monomer. </p>
 
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<h> Fluorescein Standard Curve</h>
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&#8226; Length & Fit : Intermolecular &#8226;
  
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Next, we took advantage of the fact that our hexamers act as the building blocks for our micro compartments. By using this same approximation of difference in length between the cis and trans conformation of our residue, we applied the same logic as we did with intramolecular spaces, now between hexamers. First we used the<a href="http://zdock.umassmed.edu/"> ZDOCK</a> protein docking program made available by the University of Massachusetts. </br></br>
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This program uses protein folding techniques to predict how two proteins will dock with each other, given various parameters. By using this program to dock two of our hexemeric proteins together, we are able to get a rough approximation of how our hexamers might bind. One of these predicted crystallizations is shown below.
  
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<h>ABS600 uM Fluorescein Data</h>
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Applying the same logic as we did with the intramolecular modeling, we can now find residues that fit the same parameters, except this time, the distances will be between residues on separate hexamers. We can see this applied on our two hexamers. We can see a measured distance of <b> 6.7 Angstroms </b> between our two residues, showing that this would be a suitable site for point mutation.</p>
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Latest revision as of 00:23, 16 November 2017

• Modeling •

In order to increase our efficiency, while saving time and resources, the team undertook various techniques in molecular modeling in order to discern the best places to perform point mutations. These three separate mechanisms are discussed in detail below. The 14 mutations that we eventually decided on can be seen here in red. Five of these mutations have been carried over from last year, along with nine new mutations added with this year's project.

• Length & Fit : Intramolecular •

One of the first approaches that we took in modeling was the knowledge if the approximate difference in length of our AzoPhe residue between its cis and trans conformation. We can see the individual monomers of our hexameric protein in here.

If we found individual residues that were positioned between individual monomers such that the distance between the two were greater than 6 Angstroms (the overall length of our residue in its cis conformation, and less than 13 Angstroms (the overall length of our residue in the trans conformation), then we can reason that our hexamers will be unable to pack once being activated at this point, leaving us to believe that this would be a good position for a point mutation.

As an example, we can see how this works for one of our point mutations. We see that when we zoom in, that there is a measured distance of 7.1 Angstroms between our residue of interest and the other monomer.

• Length & Fit : Intermolecular •

Next, we took advantage of the fact that our hexamers act as the building blocks for our micro compartments. By using this same approximation of difference in length between the cis and trans conformation of our residue, we applied the same logic as we did with intramolecular spaces, now between hexamers. First we used the ZDOCK protein docking program made available by the University of Massachusetts.

This program uses protein folding techniques to predict how two proteins will dock with each other, given various parameters. By using this program to dock two of our hexemeric proteins together, we are able to get a rough approximation of how our hexamers might bind. One of these predicted crystallizations is shown below.

Applying the same logic as we did with the intramolecular modeling, we can now find residues that fit the same parameters, except this time, the distances will be between residues on separate hexamers. We can see this applied on our two hexamers. We can see a measured distance of 6.7 Angstroms between our two residues, showing that this would be a suitable site for point mutation.