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From 2011.igem.org

Project Overview

'E Col Transporter'

Overview

This project is destined to generate several E. Coli strains which will cooperate with each other to move forth and back and transport any desired protein along a gradient. Module composition: binding, releasing, transition, movement

Binding

SH3 domain has high affinity for proline-rich peptides. The small size and high affinity are ideal for our carrier design. In our experiment, we used a short peptide with a Kd of 3.67μM (Y. Jacquot, et al., 2007) as the binding motif and planned to transport protein substrates with this peptide sequence.

This part comprises expression of three proteins, namely, OmpA-SH3 protein, which functions as the binding vehicle, OmpA-mCherry protein, which functions as the positive control, Proline-rich containing mCherry protein, which functions as the binding substrate.

Releasing

It’s difficult to release the substrate from strong binding, and hence protease is called into play. HIV-protease is readily available and its high efficiency, low molecular weight and high specificity constitutes fine candidate. We only need to add the substrate sequence between OmpA and SH3 sequence and the protease will cut off the binding cassette. In order to segregate the action, we’ll generate a single bacteria strain expressing the protease.

Transition

We need to shift between two states, the binding state and the releasing state. This molecular device is well-known for its lag in phase change, which is well adapted for our application. The lag during the phase transition is appropriate for movement and transport.

Movement

Kinase system is well utilized in E. Coli movement control. We’ll again use LVA tagged kinase to drive E. Coli towards the target.