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

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          loxP site sequence: ATAACTTCGTATA–GCATACAT–TATACGAAGTTAT  
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    loxP site sequence: ATAACTTCGTATA–GCATACAT–TATACGAAGTTAT  
The recombination event is dependent on the location and relative orientation of the loxP sites, which can be in cis (same DNA strand) or trans (different DNA strands) position. The spacer sequence (8bp) found in each loxP site is also important for the outcome of the recombination process. It gives the orientation of one site relative to the other when both are located on the same  strand. When two loxP sites are oriented in the same direction the DNA between them will be excised as a circular loop. However, when these two loxP sites are in opposite directions, the DNA between them will be inverted (5) (Fig. 1). The enzyme requires no additional cofactors (such as ATP) or accessory proteins for its function (1).
The recombination event is dependent on the location and relative orientation of the loxP sites, which can be in cis (same DNA strand) or trans (different DNA strands) position. The spacer sequence (8bp) found in each loxP site is also important for the outcome of the recombination process. It gives the orientation of one site relative to the other when both are located on the same  strand. When two loxP sites are oriented in the same direction the DNA between them will be excised as a circular loop. However, when these two loxP sites are in opposite directions, the DNA between them will be inverted (5) (Fig. 1). The enzyme requires no additional cofactors (such as ATP) or accessory proteins for its function (1).
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig2plugplay.JPG | caption= Fig 2. 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 }}
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig2plugplay.JPG | caption= Fig. 2. 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 }}
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The first recombination event consists in circularizing 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. This step results in a  circular fragment comprising the target gene and one lox61 recognition site newly created (Fig. 3), it also produce an 8bp loxP fragment that is released.
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The first recombination event consists in circularizing the linear 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. This step results in a  circular fragment comprising the target gene and one lox61 recognition site newly created (Fig. 3), it also produce an 8bp loxP fragment that is released.
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The second recombination event 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), as an integrative recombination. It results in a bigger plasmid with the target gene located in a context ready to be expressed, flanked by one loxP site upstream and one double mutated site downstream. which reduces the chance of the fragment to be subsequently excised (Fig 3).
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The second recombination event 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), as an integrative recombination. It results in a bigger plasmid with the target gene located in a context ready to be expressed, flanked by one loxP site upstream and one double mutated site downstream. which reduces the chance of the fragment to be subsequently excised (Fig. 3).
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=fig3plugplay.JPG | caption= Fig.3 ORF circularization and insertion in Plug&Play plasmid | size=600px }}
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Pplay_fig.jpeg | caption= Fig.3. ORF circularization and insertion in Plug&Play plasmid | size=600px }}
This project aims to accomplish the expression of any gene in a two-step process: a PCR reaction and a bacterial transformation. The linear DNA from the PCR is directly inserted into electrocompetent ''E. coli'' cells (e.g. BL21(DE3)) already harboring the Plug&play plasmid and expressing Cre recombinase for the circularization and the integration of the target gene. Mathematical modeling performed to predict rates of linear DNA degradation, integration and circularization showed the viability of the project even before beginning to work in the bench.
This project aims to accomplish the expression of any gene in a two-step process: a PCR reaction and a bacterial transformation. The linear DNA from the PCR is directly inserted into electrocompetent ''E. coli'' cells (e.g. BL21(DE3)) already harboring the Plug&play plasmid and expressing Cre recombinase for the circularization and the integration of the target gene. Mathematical modeling performed to predict rates of linear DNA degradation, integration and circularization showed the viability of the project even before beginning to work in the bench.
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7. VAN DUYNE G (2001). "A Structural View of Cre-loxP Site Specific Recombination". Annual Reviews Biophysics Biomolcular Structures 30: 87–104.
7. VAN DUYNE G (2001). "A Structural View of Cre-loxP Site Specific Recombination". Annual Reviews Biophysics Biomolcular Structures 30: 87–104.
8. VOZIYANOV Y, PATHANIA S, JAYARAM M. 1999. A general model for sites pecific recombination by the integrase family recombinases. Nucleic Acids Res 27:930–941
8. VOZIYANOV Y, PATHANIA S, JAYARAM M. 1999. A general model for sites pecific recombination by the integrase family recombinases. Nucleic Acids Res 27:930–941
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Latest revision as of 22:20, 25 September 2012