Team:Dundee/Modeling/bdsf

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               <h1>Modelling BDSF</h1>  
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             <p class="lead">What we did</p>
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             <p class="lead">Prediction of Cross-Talking Kinase</p>
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             <li class="list-group-item"><a href="#0" class="">Initial planning and cloning strategy</a>
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             <li class="list-group-item"><a href="#0" class="">Objectives</a>
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             <li class="list-group-item"><a href="#1" class="">Building the PQS sensor</a>
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             <li class="list-group-item"><a href="#1" class="">BDSF-Induced Phosphorylation of BCAM0228[P] Mediates GFP Expression</a>
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             <li class="list-group-item"><a href="#2" class="">Characterisation</a>  
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             <li class="list-group-item"><a href="#2" class="">BCAM0228 is Phosphorylated by Unknown Compound</a>
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            <li class="list-group-item"><a href="#3" class="">Conclusion</a>  
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To investigate why GFP expression was high in the absence of signal in our engineered BDSF system.
To investigate why GFP expression was high in the absence of signal in our engineered BDSF system.
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<h2>BDSF-Induced Phosphorylation of BCAM0228[P] Mediates GFP Expression</h2>
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<h2 id="1">BDSF-Induced Phosphorylation of BCAM0228[P] Mediates GFP Expression</h2>
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We constructed models similar to those used in the PQS system to investigate the signal-response behaviour of the BDSF system. Our results verified what we had expected to happen. Phosphorylation of BCAM0228 is induced by BDSF binding to a cell receptor.  BCAM0228[P] then activated the expression of GFP through the engineered <i>cvld</i> promoter. In the absence of a BDSF signal, BCAM0228 remained in its unphosphorylated form and GFP expression was repressed.  Increasing the signal induced an increased GFP response (Fig 1).
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We constructed models<sup>1</sup> similar to those used in the PQS system to investigate the signal-response behaviour of the BDSF system. Using the parameters defined in table 1, our results<sup>2,3</sup> verified what we had expected to happen. Phosphorylation of BCAM0228 is induced by BDSF binding to a cell receptor.  BCAM0228[P] then activated the expression of GFP through the engineered <i>cbld</i> promoter. In the absence of a BDSF signal, BCAM0228 remained in its unphosphorylated form and GFP expression was repressed.  Increasing the signal induced an increased GFP response (Fig 1).
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<h2>BCAM0228 is Phosphorylated by Unknown Compound</h2>
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<h2 id="2">BCAM0228 is Phosphorylated by Unknown Compound</h2>
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Our experimental results revealed that in the absence of BDSF, the cblD promoter was active in our engineered system and hence GFP expression upregulated.  
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Our experimental results revealed that in the absence of BDSF, the <i>cblD</i> promoter was active in our engineered system and hence GFP expression upregulated.  
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            <img data-alt-src="https://static.igem.org/mediawiki/2014/1/1c/BDSF_fig2.png"
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From our system of equations we know that the expression of GFP is dependant on the levels of BCAM0228[P]. We hypothesised that one possible mechanism by which our cell could be expressing GFP in the absence of signal was by the presence of BCAM0228[P] independent of BDSF-binding mediated phosphorylation. This suggests that an unknown compound could be phosphorylating BCAM0228 in our engineered cells.  
From our system of equations we know that the expression of GFP is dependant on the levels of BCAM0228[P]. We hypothesised that one possible mechanism by which our cell could be expressing GFP in the absence of signal was by the presence of BCAM0228[P] independent of BDSF-binding mediated phosphorylation. This suggests that an unknown compound could be phosphorylating BCAM0228 in our engineered cells.  
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Comparing Fig 2 to Fig 1, it is clear that the model predicts that the expression of GFP is higher when BCAM0228 is being constitutively phosphorylated than in the “normal” case. Moreover, we are also able to conclude that the model predicts that the presence of BDSF has very little effect on the overall expression of GFP when the constitutive phosphorylation process is operating.  
Comparing Fig 2 to Fig 1, it is clear that the model predicts that the expression of GFP is higher when BCAM0228 is being constitutively phosphorylated than in the “normal” case. Moreover, we are also able to conclude that the model predicts that the presence of BDSF has very little effect on the overall expression of GFP when the constitutive phosphorylation process is operating.  
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<sup>1</sup><a href="https://static.igem.org/mediawiki/2014/0/0b/Appendix_2_-_BDSF.pdf">https://static.igem.org/mediawiki/2014/0/0b/Appendix_2_-_BDSF.pdf</a><br>
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<sup>2</sup><a href="https://static.igem.org/mediawiki/2014/2/28/Final_setup_bdsf.m">https://static.igem.org/mediawiki/2014/2/28/Final_setup_bdsf.m</a><br>
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<sup>3</sup><a href="https://static.igem.org/mediawiki/2014/4/4f/Final_solver_bdsf.m">https://static.igem.org/mediawiki/2014/4/4f/Final_solver_bdsf.m</a><br>
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<sup>4</sup>Leake, M. et al. (2008)  Proceedings of the National Academy of Sciences USA 105, 15376-15381.<br>
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<sup>5</sup>Andrea J. Twigg & David Sherratt (1980)  Nature 283, 216 - 218.
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<a href="" class="btn btn-default toLesson">Previous lesson</a>   
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Latest revision as of 22:23, 17 October 2014

Dundee 2014

Dundee 2014

Modelling BDSF

Prediction of Cross-Talking Kinase

Objectives

To investigate why GFP expression was high in the absence of signal in our engineered BDSF system.

BDSF-Induced Phosphorylation of BCAM0228[P] Mediates GFP Expression

We constructed models1 similar to those used in the PQS system to investigate the signal-response behaviour of the BDSF system. Using the parameters defined in table 1, our results2,3 verified what we had expected to happen. Phosphorylation of BCAM0228 is induced by BDSF binding to a cell receptor. BCAM0228[P] then activated the expression of GFP through the engineered cbld promoter. In the absence of a BDSF signal, BCAM0228 remained in its unphosphorylated form and GFP expression was repressed. Increasing the signal induced an increased GFP response (Fig 1).



BCAM0228 is Phosphorylated by Unknown Compound

Our experimental results revealed that in the absence of BDSF, the cblD promoter was active in our engineered system and hence GFP expression upregulated.



From our system of equations we know that the expression of GFP is dependant on the levels of BCAM0228[P]. We hypothesised that one possible mechanism by which our cell could be expressing GFP in the absence of signal was by the presence of BCAM0228[P] independent of BDSF-binding mediated phosphorylation. This suggests that an unknown compound could be phosphorylating BCAM0228 in our engineered cells.

Comparing Fig 2 to Fig 1, it is clear that the model predicts that the expression of GFP is higher when BCAM0228 is being constitutively phosphorylated than in the “normal” case. Moreover, we are also able to conclude that the model predicts that the presence of BDSF has very little effect on the overall expression of GFP when the constitutive phosphorylation process is operating.


Conclusions

Our models predict that an as yet unknown agent phosphorylates BCAM0228 in the absence of a BDSF signal and that in fact the level of the BDSF signal has little quantifiable effect on GFP expression in our engineered cells.

References

1https://static.igem.org/mediawiki/2014/0/0b/Appendix_2_-_BDSF.pdf
2https://static.igem.org/mediawiki/2014/2/28/Final_setup_bdsf.m
3https://static.igem.org/mediawiki/2014/4/4f/Final_solver_bdsf.m
4Leake, M. et al. (2008) Proceedings of the National Academy of Sciences USA 105, 15376-15381.
5Andrea J. Twigg & David Sherratt (1980) Nature 283, 216 - 218.