Team:Paris Bettencourt/Restriction Enzyme

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Overview

Our group was responsible for designing self-plasmid digestion system. This synthetic system allows to digest plasmids into linear parts of DNA which afterwards could be degraded by Colicin.

Objectives

1. To find appropriate restriction enzymes which have to match the next properties:
   • In the E.Coli genome there is no restriction sites of a choosen restriction enzyme;
   • It has to have high specifity;
   • It have to works in wide range of different conditions (pH, T°C, etc)
2. Choose very strong promoter to regulate restriction enzyme expression;
3. To clone circuits with different combinations of choosen restriction enzymes and promoters.
4. Mesure degradation efficiency for each circuit.
5. Based on the best combinantion design self-disruption plasmid.

Design

On a role of restriction ensime we found two candidates:

1. Fse I - restriction endonucleases which recognize 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The most important to methion that it has the lowest number of restriction sites in E.Coli genome: only 4 copies. We decided to use MAGE to remove it from chromosome (see for more detiles), but after MAGE did not have the expected yield, we decided to stop it and works only wuth the next candidate.
2. I-SceI

Experiments and results

Characterisation of X

Experimental setup

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Results

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Testing of the system

Experimental setup

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Results

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