Team:USP-UNESP-Brazil/Plasmid Plug n Play/Modeling

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<p>Although we did not consider the mutated loxP, we have some considerations about it. The insertion reaction is favored over the excision reaction by roughly fivefold using mutated recombination, when using CRE recombinases <span class="citation"></span>. This occurs because the double mutated loxP has a very low affinity for the CRE monomers. So, an intuitive conclusion is that the combination we chose may optimize the insertion of the ORF in the Plug&Play plasmid. Nevertheless, this conclusion could be false because the altered loxP demands more time in the circularization step since it has a lower association constant for CRE recombinase. This extra amount of time could be such, that the degradation of linear DNA plays a fundamental role in the process. However, as it is illustrated, in the case of CRE recombinases the degradation of linear DNA is not a fundamental variable and it may not interfere. Because of this, the combination of mutated loxP must optimize the amount of ORF inserted in the plasmid.</p>
<p>Although we did not consider the mutated loxP, we have some considerations about it. The insertion reaction is favored over the excision reaction by roughly fivefold using mutated recombination, when using CRE recombinases <span class="citation"></span>. This occurs because the double mutated loxP has a very low affinity for the CRE monomers. So, an intuitive conclusion is that the combination we chose may optimize the insertion of the ORF in the Plug&Play plasmid. Nevertheless, this conclusion could be false because the altered loxP demands more time in the circularization step since it has a lower association constant for CRE recombinase. This extra amount of time could be such, that the degradation of linear DNA plays a fundamental role in the process. However, as it is illustrated, in the case of CRE recombinases the degradation of linear DNA is not a fundamental variable and it may not interfere. Because of this, the combination of mutated loxP must optimize the amount of ORF inserted in the plasmid.</p>
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<h1 id="appendix">Appendix</h1>
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<h1 id="Appendix">Appendix</h1>
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<h2 id="Equations">Equations</h2>
<p><br /><span class="math">$  \frac{d}{dt} [S]      = k_{-1}[S_{a}] - [S] \left(k_{1}[M] + k_{d}\right) $</span><br /></p>
<p><br /><span class="math">$  \frac{d}{dt} [S]      = k_{-1}[S_{a}] - [S] \left(k_{1}[M] + k_{d}\right) $</span><br /></p>
<p><br /><span class="math">$  \frac{d}{dt} [S_{a}]  = k_{1}[S][M] + k_{-1}[S_{aa}] + k_{-2}[S_{ab}] - [S_{a}]( k_{1}[M] + k_{-1} + k_{2}[M] + k_{d} ) $</span><br /></p>
<p><br /><span class="math">$  \frac{d}{dt} [S_{a}]  = k_{1}[S][M] + k_{-1}[S_{aa}] + k_{-2}[S_{ab}] - [S_{a}]( k_{1}[M] + k_{-1} + k_{2}[M] + k_{d} ) $</span><br /></p>

Revision as of 17:13, 25 September 2012