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− | <h2 style="width: | + | <h2 style="width:70%;margin-left:15%">Linker Development</h2> |
− | <h5 style="width: | + | <h5 style="width:70%;margin-left:15%">Summary</h5> |
− | <p style="width: | + | <p style="width:70%;margin-left:15%">As a part of our construct it is necessary to build linkers to connect the intein halves with the target receptor. The challenge in |
developing linkers for the system is that they must be of a specific length that will allow association of the intein halves in the | developing linkers for the system is that they must be of a specific length that will allow association of the intein halves in the | ||
bound conformation of the receptor but will not allow association of the intein halves in the unbound conformation of the receptor. | 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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attempts at creating linkers were made.</p> | attempts at creating linkers were made.</p> | ||
− | <h5 style="width: | + | <h5 style="width:70%;margin-left:15%">Approaches</h5> |
− | <h6 style="width: | + | <h6 style="width:70%;margin-left:15%">Initial Design</h6> |
− | <p style="width: | + | <p style="width:70%;margin-left:15%">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 |
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 | 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 | ||
used in the paper. This approach was flawed however as it overlooked the differences in the receptor conformation changes.</p> | used in the paper. This approach was flawed however as it overlooked the differences in the receptor conformation changes.</p> | ||
− | <figure style="width: | + | <figure style="width:70%;margin-left:15%"> |
<img src="https://static.igem.org/mediawiki/2018/6/62/T--Queens_Canada--PyMOLEstrogenLinker.jpg" alt='estrogenlinker'/> | <img src="https://static.igem.org/mediawiki/2018/6/62/T--Queens_Canada--PyMOLEstrogenLinker.jpg" alt='estrogenlinker'/> | ||
<figcaption>This image shows the system modelled in PyMOL without the extein using the linkers from the Estrogen paper the design is inspired | <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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</figure> | </figure> | ||
− | <h6 style="width: | + | <h6 style="width:70%;margin-left:15%">Uniformed Design</h6> |
− | <p style="width: | + | <p style="width:70%;margin-left:15%">While testing the initial design it was also decided to test other linkers at the same time. These linkers were generated by using common |
synthetic linkers, found through research, and testing to see if an association event was viable by modelling the system on PyMOL. This | synthetic linkers, found through research, and testing to see if an association event was viable by modelling the system on PyMOL. This | ||
approach generated linkers that were too long as we were focussed on making linkers long enough for the interaction but forgot about the | approach generated linkers that were too long as we were focussed on making linkers long enough for the interaction but forgot about the | ||
need for the linkers to prevent interaction in the unbound conformation.</p> | need for the linkers to prevent interaction in the unbound conformation.</p> | ||
− | <figure style="width: | + | <figure style="width:70%;margin-left:15%"> |
<img src="https://static.igem.org/mediawiki/2018/8/8d/T--Queens_Canada--PyMOLPolyglycineLinker.jpg" alt='polyglycinelinker'/> | <img src="https://static.igem.org/mediawiki/2018/8/8d/T--Queens_Canada--PyMOLPolyglycineLinker.jpg" alt='polyglycinelinker'/> | ||
<figcaption >This image shows the system modelled in PyMOL without the extein using poly-glycine linkers. The glucocorticoid receptor is in its | <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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</figure> | </figure> | ||
− | <h6 style="width: | + | <h6 style="width:70%;margin-left:15%">Informed Design</h6> |
− | <p style="width: | + | <p style="width:70%;margin-left:15%">New linkers were developed by discussing with Professors at Queen’s University for advice on linker design. As such were made aware of more |
variables to consider when designing the linker including flexibility and secondary structure.</p> | variables to consider when designing the linker including flexibility and secondary structure.</p> | ||
− | <figure style="width: | + | <figure style="width:70%;margin-left:15%"> |
<img src="https://static.igem.org/mediawiki/2018/3/39/T--Queens_Canada--PyMOLFlexibilityLinker.jpg" alt='flexibilitylinker'/> | <img src="https://static.igem.org/mediawiki/2018/3/39/T--Queens_Canada--PyMOLFlexibilityLinker.jpg" alt='flexibilitylinker'/> | ||
<figcaption >This image shows the system modelled in PyMOL without the extein using linkers that consider flexibility. These linkers include some | <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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</figure> | </figure> | ||
− | <h6 style="width: | + | <h6 style="width:70%;margin-left:15%">Modelled Design</h6> |
− | <p style="width: | + | <p style="width:70%;margin-left:15%">To try to make more informed linkers we talked with another Professor at Queen’s University. From this discussion we discussed the shortest |
path around the molecule that would be ideal for the association event. We were also provided with web databases that contain the structure and | path around the molecule that would be ideal for the association event. We were also provided with web databases that contain the structure and | ||
information for synthetic and natural linkers. In PyMOL the angstrom distances on the newly chosen path were determined and linkers with the | information for synthetic and natural linkers. In PyMOL the angstrom distances on the newly chosen path were determined and linkers with the | ||
appropriate length and flexibility were made.</p> | appropriate length and flexibility were made.</p> | ||
− | <figure style="width: | + | <figure style="width:70%;margin-left:15%"> |
<img src="https://static.igem.org/mediawiki/2018/b/b6/T--Queens_Canada--PyMOLNoLinker.jpg" alt='nolinker'/> | <img src="https://static.igem.org/mediawiki/2018/b/b6/T--Queens_Canada--PyMOLNoLinker.jpg" alt='nolinker'/> | ||
<figcaption>This image shows the system modelled in PyMOL without the extein or linkers. The glucocorticoid receptor is in its agonist bound | <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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</figure> | </figure> | ||
− | <a href="https://2018.igem.org/Team:Queens_Canada/Linker_Software"><h6 style="width: | + | <a href="https://2018.igem.org/Team:Queens_Canada/Linker_Software"><h6 style="width:70%;margin-left:15%">Software Design (click here to find out more!)</h6></a> |
− | <p style="width: | + | <p style="width:70%;margin-left:15%">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 |
user and design a linker that will span the space.</p> | user and design a linker that will span the space.</p> | ||
<footer style="background-color: #212121;height:90px "> | <footer style="background-color: #212121;height:90px "> |
Revision as of 22:50, 18 September 2018
Linker Development
Summary
As a part of our construct it is necessary to build linkers to connect the intein halves with the target receptor. The challenge in developing linkers for the system is that they must be of a specific length that will allow association of the intein halves in the bound conformation of the receptor but will not allow association of the intein halves in the unbound conformation of the receptor. In addition, the flexibility of the linkers must be adjusted for the same purpose. In comparison, the scientific paper from which our project draws its inspiration has a much simpler time developing linkers as the change in confirmation of their chosen receptor created a significantly larger change in distance compared to the receptor that we are looking at. As the project progressed many different attempts at creating linkers were made.
Approaches
Initial Design
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 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 used in the paper. This approach was flawed however as it overlooked the differences in the receptor conformation changes.
Uniformed Design
While testing the initial design it was also decided to test other linkers at the same time. These linkers were generated by using common synthetic linkers, found through research, and testing to see if an association event was viable by modelling the system on PyMOL. This approach generated linkers that were too long as we were focussed on making linkers long enough for the interaction but forgot about the need for the linkers to prevent interaction in the unbound conformation.
Informed Design
New linkers were developed by discussing with Professors at Queen’s University for advice on linker design. As such were made aware of more variables to consider when designing the linker including flexibility and secondary structure.
Modelled Design
To try to make more informed linkers we talked with another Professor at Queen’s University. From this discussion we discussed the shortest path around the molecule that would be ideal for the association event. We were also provided with web databases that contain the structure and information for synthetic and natural linkers. In PyMOL the angstrom distances on the newly chosen path were determined and linkers with the appropriate length and flexibility were made.
Software Design (click here to find out more!)
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 user and design a linker that will span the space.