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

From 2012.igem.org

(Difference between revisions)
Line 15: Line 15:
However, insertion of a circular DNA carrying a loxP into a loxP site on a chromosome (integrative recombination) is quite inefficient because unimolecular reactions are kinetically favored over bimolecular reactions, causing that the inserted DNA will often be excised (6) (Fig. 2a). To tackle this problem we have used loxP mutant sites (2). These sites help to maintain the inserted DNA in the chromosome (Fig. 2b).In our project we used one loxP site and two loxP mutant sites (lox71 and lox66) (Fig. 2c).
However, insertion of a circular DNA carrying a loxP into a loxP site on a chromosome (integrative recombination) is quite inefficient because unimolecular reactions are kinetically favored over bimolecular reactions, causing that the inserted DNA will often be excised (6) (Fig. 2a). To tackle this problem we have used loxP mutant sites (2). These sites help to maintain the inserted DNA in the chromosome (Fig. 2b).In our project we used one loxP site and two loxP mutant sites (lox71 and lox66) (Fig. 2c).
-
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig2plugplay.jpg | caption=Illustration of Cre/loxP and Cre/LE- and RE-mutant lox system. Black regions of the triangles represent sites at which nucleotide sequence changes occurred. a Recombination between loxP sites. b Recombination between a LE mutant lox71 and a RE mutant lox66 produces a loxP and a LE + RE mutant lox. c Gray boxes indicate sites at which nucleotide sequence changes occurred (6). | size=600px }}
+
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig2plugplay.JPG | caption=Illustration of Cre/loxP and Cre/LE- and RE-mutant lox system. Black regions of the triangles represent sites at which nucleotide sequence changes occurred. a Recombination between loxP sites. b Recombination between a LE mutant lox71 and a RE mutant lox66 produces a loxP and a LE + RE mutant lox. c Gray boxes indicate sites at which nucleotide sequence changes occurred (6). | size=600px }}
The first recombination consists in a circularization of the linearized PCR product (target-gene flanked by one loxP site upstream and one lox66 site downstream, both in the same orientation) in order to form a plasmid. The result of this step is one 8bp loxP fragment that is released and one circular fragment with the target gene and one lox61 newly created (Fig. 3).
The first recombination consists in a circularization of the linearized PCR product (target-gene flanked by one loxP site upstream and one lox66 site downstream, both in the same orientation) in order to form a plasmid. The result of this step is one 8bp loxP fragment that is released and one circular fragment with the target gene and one lox61 newly created (Fig. 3).
The second recombination occurs between the circularized PCR product and the Plug&Play plasmid (containing the Cre recombinase gene, a T7 promoter, a lox71 site, a stop site and a ampicillin resistance gene), in an integrative recombination. As result, we will have one bigger plasmid with the target gene flanked by one loxP site and one double mutated site, reducing the chance of the fragment to be excised and locating the gene in a context ready to express it (Fig 3).
The second recombination occurs between the circularized PCR product and the Plug&Play plasmid (containing the Cre recombinase gene, a T7 promoter, a lox71 site, a stop site and a ampicillin resistance gene), in an integrative recombination. As result, we will have one bigger plasmid with the target gene flanked by one loxP site and one double mutated site, reducing the chance of the fragment to be excised and locating the gene in a context ready to express it (Fig 3).
-
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig3plugplay.jpg | caption= Fig.3 | size=600px }}
+
{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig3plugplay.JPG | caption= Fig.3 | size=600px }}
This project aims to reduce the expression of any gene to two steps; PCR and transformation. The linear DNA, from the PCR, is inserted in electro-competent cells (BL21(DE3)), the electro-competent cells already have the plug&play plasmid producing Cre recombinase for the circularization and the integration. Issues like the linear DNA degradation, integration and circularization rates were explored using mathematical modeling, which showed the viability of the project even before beginning to work in the bench.  
This project aims to reduce the expression of any gene to two steps; PCR and transformation. The linear DNA, from the PCR, is inserted in electro-competent cells (BL21(DE3)), the electro-competent cells already have the plug&play plasmid producing Cre recombinase for the circularization and the integration. Issues like the linear DNA degradation, integration and circularization rates were explored using mathematical modeling, which showed the viability of the project even before beginning to work in the bench.  

Revision as of 19:02, 21 September 2012