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

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<p>In order to identify differences between FLP and CRE, we compared the two enzymes using two analyses. Our results point to an obvious choice for the CRE-lox recombination system since it is less affected by DNA degradation and improves the insertion of the ORF compared with FLP-FRT system.</p>
<p>In order to identify differences between FLP and CRE, we compared the two enzymes using two analyses. Our results point to an obvious choice for the CRE-lox recombination system since it is less affected by DNA degradation and improves the insertion of the ORF compared with FLP-FRT system.</p>
<p>In our model we have considered all lox sites as loxP. However, there are mutated loxP and a combination of them can improve the insertion of the target gene (ORF) [4]. We have chosen to use lox66 and lox71 in our experimental design. Nevertheless, we did not introduce the lox66 and lox71 in the model for two main reasons: there are no references about the values of rate constants for altered loxP and we prefer to keep the simplicity and clarity of the model. In order to take these variables in consideration, it would be necessary to use more equations and extra hypothesis.</p>
<p>In our model we have considered all lox sites as loxP. However, there are mutated loxP and a combination of them can improve the insertion of the target gene (ORF) [4]. We have chosen to use lox66 and lox71 in our experimental design. Nevertheless, we did not introduce the lox66 and lox71 in the model for two main reasons: there are no references about the values of rate constants for altered loxP and we prefer to keep the simplicity and clarity of the model. In order to take these variables in consideration, it would be necessary to use more equations and extra hypothesis.</p>
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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 [4]. 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 Fig. 3, in the case of CRE recombinases, the degradation of linear DNA is not a fundamental variable and it may not interfere. Because of that, the combination of mutated loxP must optimize the amount of ORF inserted in the plasmid.</p>
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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 [4]. 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 Fig. 3, in the case of CRE recombinases, the degradation of linear DNA is not a fundamental variable and it may not interfere. Because of that, we may conclude that the combination of mutated loxP must optimize the amount of ORF inserted in the plasmid.</p>
<h1 id="Appendix">Appendix</h1>
<h1 id="Appendix">Appendix</h1>

Revision as of 12:12, 26 September 2012