Team:Paris/Transduction overview2 strategy

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Contents 1 Overview 1.1 B. Our Strategy 1.1.1 B.1 The Fec operon 1.1.2 B.2 The trick TCS

Overview

• Introduction
• A. Transduction
  • A.1 ABC transporters
  • A.2 Two Component System
• B. Our strategy
  • B.1 The Fec operon
  • B.2 The trick TCS
• C. Construction

B. Our Strategy

B.1 The Fec operon

Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA, moreover some mutant can also have a constitutive expression of FecABCD .

The plan would be to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system.

We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence.

problems : the message is unidirectionnal and unrepeatable. It would just be a proof of principle that a vesicle-mediated controlled communication is possible.

B.2 The trick TCS

We could ,with the help of Alfonso Jaramillo, design a synthetic PBP which could detect the substrate we choosed and activate a specific HK. The previous work done by Valencia in 2006 was to design vanilin-sensitive PBP and a network for a graduated response whereas we just need a proteic sensitive PBP and a binary type of response.

Even if the idea of creating a synthethic protein was very attractive, it would have been difficult to have concrete result in just 3 months.