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Team:EPF-Lausanne/Our Project/Plasmids strategy
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{{:Team:EPF-Lausanne/Templates/Header|title=Plasmids strategy}} | {{:Team:EPF-Lausanne/Templates/Header|title=Plasmids strategy}} | ||
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We are building a system containing TetR and LacI, that control the expression of a selection gene. The sequential use of these two repressors allows us to have a direct activation of the selection gene when TetR binds its recognition sequence. | We are building a system containing TetR and LacI, that control the expression of a selection gene. The sequential use of these two repressors allows us to have a direct activation of the selection gene when TetR binds its recognition sequence. | ||
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* in vitro: We can test the binding of TetR directly on plates by measuring RFP fluorescence | * in vitro: We can test the binding of TetR directly on plates by measuring RFP fluorescence | ||
* in vivo: The cells will grow on chemostat chips. If TetR binds to the recogintion sequence, the cells will lyse and we will be able to recover the DNA | * in vivo: The cells will grow on chemostat chips. If TetR binds to the recogintion sequence, the cells will lyse and we will be able to recover the DNA | ||
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We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this: | We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this: | ||
| - | * | + | * The '''TetR plasmid''' containing TetR under a constitutive promoter |
| - | * | + | * The '''reporter plasmid''' containing a reporter gene (either RFP or a lysis device) and a LacI inverter for tetR. |
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[[File:EPFL_Plasmids.png|700 px]] | [[File:EPFL_Plasmids.png|700 px]] | ||
Revision as of 16:07, 21 July 2011
Plasmids strategy
We are building a system containing TetR and LacI, that control the expression of a selection gene. The sequential use of these two repressors allows us to have a direct activation of the selection gene when TetR binds its recognition sequence.
.png)
The selection gene is either RFP or a lysis cassette. Having both constructs will allow us to select at two levels: in vivo and in vitro:
- in vitro: We can test the binding of TetR directly on plates by measuring RFP fluorescence
- in vivo: The cells will grow on chemostat chips. If TetR binds to the recogintion sequence, the cells will lyse and we will be able to recover the DNA
We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this:
- The TetR plasmid containing TetR under a constitutive promoter
- The reporter plasmid containing a reporter gene (either RFP or a lysis device) and a LacI inverter for tetR.
.png)
