Difference between revisions of "Team:Queens Canada/Linker Software"

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<h2>Linker Software Summary</h2>
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<h2>Linker Software Summary</h2>
  
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<h5>Summary</h5>
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<h5>Summary</h5>
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<p>The software being developed is designed to connect two selected points on the molecule by finding the  
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<p>The software being developed is designed to connect two selected points on the molecule by finding the  
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shortest path that connects them without interfering with the rest of the molecule. This is done by first  
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shortest path that connects them without interfering with the rest of the molecule. This is done by first  
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finding the shortest path at a specified resolution using an extended version of Dijkstra’s Algorithm.  
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finding the shortest path at a specified resolution using an extended version of Dijkstra’s Algorithm.  
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After the shortest path has been found linkers are designed to suit the path found. The method used to  
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After the shortest path has been found linkers are designed to suit the path found. The method used to  
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generate the linkers has yet to been determined. This software will be usable through the PyMOL software  
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generate the linkers has yet to been determined. This software will be usable through the PyMOL software  
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to account for visual representation and interaction with the molecule of choice.</p>
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to account for visual representation and interaction with the molecule of choice.</p>
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<h5>File Description</h5>
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<h5>File Description</h5>
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<p><em>Main File</em><br></p>
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<p><em>Main File</em><br></p>
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<p>The main file interfaces with PyMOL allowing for user inputs to be used in the linker software.
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<p>The main file interfaces with PyMOL allowing for user inputs to be used in the linker software.
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In addition, this file processes the inputs and manages all the function calls that coordinate the creation of the linkers.</p>
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In addition, this file processes the inputs and manages all the function calls that coordinate the creation of the linkers.</p>
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<figure>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/6/67/T--Queens_Canada--PyMOL.jpg" alt='pymol'/>
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<img src="https://static.igem.org/mediawiki/2018/6/67/T--Queens_Canada--PyMOL.jpg" alt='pymol'/>
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<figcaption>An image of the PyMOL model of the molecule used for initial testing and demonstration.</figcaption>
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<figcaption>An image of the PyMOL model of the molecule used for initial testing and demonstration.</figcaption>
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</figure>
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</figure>
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<br><p><em>Ellipsoid Fit</em><br></p>
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<br><p><em>Ellipsoid Fit</em><br></p>
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<p>The ellipsoid fit module finds the tightest fitting ellipsoid to a set of points that represent the
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<p>The ellipsoid fit module finds the tightest fitting ellipsoid to a set of points that represent the
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atoms that compose the molecule. This is done using an algorithm that is based on the Khachiyan Algorithm. This module returns the  
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atoms that compose the molecule. This is done using an algorithm that is based on the Khachiyan Algorithm. This module returns the  
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center, radii and rotation of the ellipsoid that encloses the points and allows for the plotting of the ellipsoid as a mesh graph.</p>
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center, radii and rotation of the ellipsoid that encloses the points and allows for the plotting of the ellipsoid as a mesh graph.</p>
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<figure>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/7/72/T--Queens_Canada--BestFitEllipsoid.jpg" alt='bestfit'/>
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<img src="https://static.igem.org/mediawiki/2018/7/72/T--Queens_Canada--BestFitEllipsoid.jpg" alt='bestfit'/>
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<figcaption>The best fitting ellipsoid to the set of points. The set of points that represent the atoms comprising  
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<figcaption>The best fitting ellipsoid to the set of points. The set of points that represent the atoms comprising  
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the molecule are shown above as black dots while the best fitting ellipsoid is represented as a blue mesh grid.</figcaption>
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the molecule are shown above as black dots while the best fitting ellipsoid is represented as a blue mesh grid.</figcaption>
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</figure>
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</figure>
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<br><p class="bigger"><em>Ellipsoid Points</em><br></p>
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<br><p class="bigger"><em>Ellipsoid Points</em><br></p>
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<p>The Ellipsoid Points algorithm generates a grid of points that are contained in an ellipsoid with a
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<p>The Ellipsoid Points algorithm generates a grid of points that are contained in an ellipsoid with a
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given spacing. To increase the efficiency of the algorithm the points from one of the eight quadrants are generated and the corresponding  
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given spacing. To increase the efficiency of the algorithm the points from one of the eight quadrants are generated and the corresponding  
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points from the other seven quadrants are calculated by including all the possible combinations of positive and negative values. Another
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points from the other seven quadrants are calculated by including all the possible combinations of positive and negative values. Another
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function in this module sets up a set of points for Dijkstra’s Algorithm by checking to see if the point is an obstacle, setting the points  
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function in this module sets up a set of points for Dijkstra’s Algorithm by checking to see if the point is an obstacle, setting the points  
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to unvisited, setting the distance to be infinite and the previous node to be an empty list.</p>
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to unvisited, setting the distance to be infinite and the previous node to be an empty list.</p>
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<figure>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/b/bd/T--Queens_Canada--RedPointsEllipsoid.jpg" alt='redpoints'/>
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<img src="https://static.igem.org/mediawiki/2018/b/bd/T--Queens_Canada--RedPointsEllipsoid.jpg" alt='redpoints'/>
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<figcaption>The red points in this image represent the point grid that is developed for later use in the path finding algorithm.</figcaption>
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<figcaption>The red points in this image represent the point grid that is developed for later use in the path finding algorithm.</figcaption>
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</figure>
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</figure>
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<br><p><em>Dijkstra R3</em><br></p>
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<br><p><em>Dijkstra R3</em><br></p>
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<p>This module implements an extended version of Dijkstra’s algorithm that allows for the shortest path through a  
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<p>This module implements an extended version of Dijkstra’s algorithm that allows for the shortest path through a  
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set of points in R3. The algorithm implements a heuristic approach using the Euclidean distance of the current point to the end of the  
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set of points in R3. The algorithm implements a heuristic approach using the Euclidean distance of the current point to the end of the  
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proposed linker site.</p>
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proposed linker site.</p>
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</div>
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</div>
  
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<!-- INFORMATION TO BE RELOCATED
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<!-- INFORMATION TO BE RELOCATED
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<div>
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<div>
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<h2>Eric's Latex files (to insert)</h2>
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<h2>Eric's Latex files (to insert)</h2>
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<p>We determined that the linkers between the GR-LBD and the inteins halves would need to be flexible as  
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<p>We determined that the linkers between the GR-LBD and the inteins halves would need to be flexible as  
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there is no direct path connecting the termini and they would require the flexibility to meet each other  
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there is no direct path connecting the termini and they would require the flexibility to meet each other  
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and produces a trans-splicing event. To determine which linker would be ideal we ran simulations of  
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and produces a trans-splicing event. To determine which linker would be ideal we ran simulations of  
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Root-mean-square deviation of atomic positions to determine optimal linker length and composition.  
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Root-mean-square deviation of atomic positions to determine optimal linker length and composition.  
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Firstly, we chose various linker designs inspired from previous work [a,b], then the entropy equations  
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Firstly, we chose various linker designs inspired from previous work [a,b], then the entropy equations  
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were run on the Queen’s University Computer Cluster for seven days by Dr. Campbell.
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were run on the Queen’s University Computer Cluster for seven days by Dr. Campbell.
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<ol type="a">
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<ol type="a">
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<li><a href="http://parts.igem.org/Protein_domains/Linker">http://parts.igem.org/Protein_domains/Linker</a></li>
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<li><a href="http://parts.igem.org/Protein_domains/Linker">http://parts.igem.org/Protein_domains/Linker</a></li>
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<li><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/</a></li>
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<li><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/</a></li>
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</ol>
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</ol>
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</p>
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</p>
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<p>Another aspect of our project we sought to model was which pacifier design would allow for optimal detection of the luminescence signal.  
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<p>Another aspect of our project we sought to model was which pacifier design would allow for optimal detection of the luminescence signal.  
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Eric modelled this through a MATLAB simulation of the “Narrow escape problem”....
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Eric modelled this through a MATLAB simulation of the “Narrow escape problem”....
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</p>
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</p>
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<p>Lastly, the analysis was modeled by…
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<p>Lastly, the analysis was modeled by…
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</p>
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</p>
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<h5>Summary</h5>
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<h5>Molecular Dynamics</h5>
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<p>As a part of our construct it is necessary to build linkers to connect the intein halves with the target receptor. The challenge in
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<p>To model our target system and to get a more realistic view on how the linkers would perform, the constructs were modelled on PyMOL with some  
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developing linkers for the system is that they must be of a specific length that will allow association of the intein halves in the
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of the initial linker designs. These models were then sent to the cluster computers here at Queen’s University to undergo a molecular dynamics  
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bound conformation of the receptor but will not allow association of the intein halves in the unbound conformation of the receptor.
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simulation. Dynamic modelling of molecular constructs allows for the molecule to simulate how it would vibrate in space.
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In addition, the flexibility of the linkers must be adjusted for the same purpose. In comparison, the scientific paper from which our
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</p>
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project draws its inspiration has a much simpler time developing linkers as the change in confirmation of their chosen receptor created
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<figure>
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a significantly larger change in distance compared to the receptor that we are looking at. As the project progressed many different
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<img src="https://static.igem.org/mediawiki/2018/6/62/T--Queens_Canada--PyMOLEstrogenLinker.jpg" alt='glucocorticoid'/>
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attempts at creating linkers were made.
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<figcaption>Linked glucocorticoid receptor and recA intein halves using the linkers from the Estrogen intein system paper.
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</p>
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</figcaption>
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</figure><br>
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<h5>Approaches - Initial Design</h5>
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<p>This allows for qualification of the linkers’ length and flexibility. By examining the proximity between the two halves of the intein we can  
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<p>The initial linkers that we wanted to test were from the scientific paper that was used for our project inspiration. As there is a high
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determine if an association event is possible. If the linkers do not appear to reach or if there appears to be excess length causing the halves  
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degree of homology between the receptor used in the paper and the receptor that we were interested in we decided to try the exact linkers
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of the inteins to overshoot each other the linker can be qualified as either too short or too long. The flexibility can be qualified by watching  
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used in the paper. This approach was flawed however as it overlooked the differences in the receptor conformation changes.
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to note how easily and often the two halves of the intein are able to come close to each other. If the halves of the intein are easily able to  
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</p>
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reach each other and do so more than once this indicates that the linker is probably at an appropriate flexibility for our purposes. If the halves  
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<figure>
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easily reach each other but also flail around more than expected the linkers are likely too flexible. Conversely if the halves do not easily come  
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<img src="https://static.igem.org/mediawiki/2018/6/62/T--Queens_Canada--PyMOLEstrogenLinker.jpg" alt='estrogenlinker'/>
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into proximity than the linker is likely too inflexible.
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<figcaption>This image shows the system modelled in PyMOL without the extein using the linkers from the Estrogen paper the design is inspired
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</p>
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by. The glucocorticoid receptor is in its agonist bound conformation, shown in green, helix 1 of the receptor is in orange and helix 12 is blue.
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-->
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The RecA intein halves are in purple and white, while the linkers are yellow.
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</figcaption>
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</figure>
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<h5>Approaches - Uniformed Design</h5>
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<p>While testing the initial design it was also decided to test other linkers at the same time. These linkers were generated by using common
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synthetic linkers, found through research, and testing to see if an association event was viable by modelling the system on PyMOL. This
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approach generated linkers that were too long as we were focussed on making linkers long enough for the interaction but forgot about the
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need for the linkers to prevent interaction in the unbound conformation.
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/8/8d/T--Queens_Canada--PyMOLPolyglycineLinker.jpg" alt='polyglycinelinker'/>
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<figcaption>This image shows the system modelled in PyMOL without the extein using poly-glycine linkers. The glucocorticoid receptor is in its
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agonist bound conformation, shown in green, helix 1 of the receptor is in orange and helix 12 is blue. The RecA intein halves are in purple and
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white, while the linkers are yellow.
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</figcaption>
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</figure>
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<h5>Approaches - Informed Design</h5>
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<p>New linkers were developed by discussing with Professors at Queen’s University for advice on linker design. As such were made aware of more
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variables to consider when designing the linker including flexibility and secondary structure.
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/3/39/T--Queens_Canada--PyMOLFlexibilityLinker.jpg" alt='flexibilitylinker'/>
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<figcaption>This image shows the system modelled in PyMOL without the extein using linkers that consider flexibility. These linkers include some
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amino acids like proline to introduce some rigidity. The glucocorticoid receptor is in its agonist bound conformation, shown in green, helix 1 of
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the receptor is in orange and helix 12 is blue. The RecA intein halves are in purple and white, while the linkers are yellow.
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</figcaption>
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</figure>
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<h5>Approaches - Modelled Design</h5>
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<p>To try to make more informed linkers we talked with another Professor at Queen’s University. From this discussion we discussed the shortest
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path around the molecule that would be ideal for the association event. We were also provided with web databases that contain the structure and
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information for synthetic and natural linkers. In PyMOL the angstrom distances on the newly chosen path were determined and linkers with the
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appropriate length and flexibility were made.
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/b/b6/T--Queens_Canada--PyMOLNoLinker.jpg" alt='nolinker'/>
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<figcaption>This image shows the system modelled in PyMOL without the extein or linkers. The glucocorticoid receptor is in its agonist bound
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conformation, shown in green, helix 1 of the receptor is in orange and helix 12 is blue. The RecA intein halves are in purple and white with the
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portions that will be attached to the linkers coloured in red. In this image the RecA halves are positioned where they would likely associate with
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each other. The angstrom distances between the RecA halves and their respective helices on the glucocorticoid receptor are shown by the yellow
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dotted lines.
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</figcaption>
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</figure>
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<h5>Approaches - Software Design</h5>
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<p>To further the work done to design linkers a software program that interacts with PyMOL is being developed to find the shortest path for the
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user and design a linker that will span the space.
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</p>
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<h5>Molecular Dynamics</h5>
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<p>To model our target system and to get a more realistic view on how the linkers would perform, the constructs were modelled on PyMOL with some  
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of the initial linker designs. These models were then sent to the cluster computers here at Queen’s University to undergo a molecular dynamics  
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simulation. Dynamic modelling of molecular constructs allows for the molecule to simulate how it would vibrate in space.
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2018/6/62/T--Queens_Canada--PyMOLEstrogenLinker.jpg" alt='glucocorticoid'/>
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<figcaption>Linked glucocorticoid receptor and recA intein halves using the linkers from the Estrogen intein system paper.
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</figcaption>
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</figure><br>
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<p>This allows for qualification of the linkers’ length and flexibility. By examining the proximity between the two halves of the intein we can  
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determine if an association event is possible. If the linkers do not appear to reach or if there appears to be excess length causing the halves  
+
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of the inteins to overshoot each other the linker can be qualified as either too short or too long. The flexibility can be qualified by watching  
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to note how easily and often the two halves of the intein are able to come close to each other. If the halves of the intein are easily able to  
+
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reach each other and do so more than once this indicates that the linker is probably at an appropriate flexibility for our purposes. If the halves  
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easily reach each other but also flail around more than expected the linkers are likely too flexible. Conversely if the halves do not easily come  
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into proximity than the linker is likely too inflexible.
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</p>
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Revision as of 18:12, 5 September 2018

Linker Software Summary

Summary

The software being developed is designed to connect two selected points on the molecule by finding the shortest path that connects them without interfering with the rest of the molecule. This is done by first finding the shortest path at a specified resolution using an extended version of Dijkstra’s Algorithm. After the shortest path has been found linkers are designed to suit the path found. The method used to generate the linkers has yet to been determined. This software will be usable through the PyMOL software to account for visual representation and interaction with the molecule of choice.

File Description

Main File

The main file interfaces with PyMOL allowing for user inputs to be used in the linker software. In addition, this file processes the inputs and manages all the function calls that coordinate the creation of the linkers.

pymol
An image of the PyMOL model of the molecule used for initial testing and demonstration.

Ellipsoid Fit

The ellipsoid fit module finds the tightest fitting ellipsoid to a set of points that represent the atoms that compose the molecule. This is done using an algorithm that is based on the Khachiyan Algorithm. This module returns the center, radii and rotation of the ellipsoid that encloses the points and allows for the plotting of the ellipsoid as a mesh graph.

bestfit
The best fitting ellipsoid to the set of points. The set of points that represent the atoms comprising the molecule are shown above as black dots while the best fitting ellipsoid is represented as a blue mesh grid.

Ellipsoid Points

The Ellipsoid Points algorithm generates a grid of points that are contained in an ellipsoid with a given spacing. To increase the efficiency of the algorithm the points from one of the eight quadrants are generated and the corresponding points from the other seven quadrants are calculated by including all the possible combinations of positive and negative values. Another function in this module sets up a set of points for Dijkstra’s Algorithm by checking to see if the point is an obstacle, setting the points to unvisited, setting the distance to be infinite and the previous node to be an empty list.

redpoints
The red points in this image represent the point grid that is developed for later use in the path finding algorithm.

Dijkstra R3

This module implements an extended version of Dijkstra’s algorithm that allows for the shortest path through a set of points in R3. The algorithm implements a heuristic approach using the Euclidean distance of the current point to the end of the proposed linker site.