Team:Paris Bettencourt/SID

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Overview

Bacteria developed mechanisms to kill other bacteria, thus reducing competition between them in the environment. Some strains of E. coli produce lethal proteins called colicins which kill other bacteria, including E. coli. Colicins are built out of three main domains which are also a point of difference among many types of colicins. First domain is responsible for binding to a receptor on a bacterial membrane, second one is responsible for translocating the protein from outside to inside of a bacteria and the third one is responsible for killing bacteria. We are interested in two types of colicins, colicin E2 and colicin D. Both of them use different binding, translocation and killing mechanisms. Colicin E2 is a DNAse and colicin D is an RNAse. [put a scheme]

We hypothesize that it is possible to use binding and translocation part of colicin as a "synthetic import domain" onto which we can fuse other proteins in order to import them into bacteria. Main aim of this part of the project is to fuse the RNAse domain from colicin D to the "synthetic import domain" of colicin E2. By doing this we will obtain a toxin that targets the same receptor and translocation mechanism as colicin E2 but kills bacteria on the level of protein synthesis by cleaving tRNA as the RNAse domain of colicin D. [link to suicide group, and say how it links to the suicide group (new toxin to use)]]. [put a scheme]

In addition to this, we fused restriction enzymes FseI and I-SceI both to the colicin E2 and colicin D "synthetic import domain". These two enzymes are used in different parts of our project [link to restriction group]. We also fused a couple of other proteins to easily test the plausibility of our "synthetic import domain" system. So far we managed to build the majority of our constructs but the experimental confirmation is still lacking. [put a scheme]

Objectives

Write here the objectives of your project

1. e2+rnased 2. e2 or d + enzymes 3. e2 or d + test stuff 4. making new import mechanism

Design

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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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