S.tar, by iGEM Technion 2016

S.Tar components

Tar Improvements and Characterization

PctA-Tar

NarX-Tar

Intein

hERα-Tar Chimera

Computational Design

S.Tar Introduction

Chemotaxis is the movement of an organism in response to an external chemical stimulus The bacterial chemotaxis system is characterized by specific responses, high sensitivity and a dynamic range (1). This system enables bacteria to sense their immediate environment and quickly adapt to changes in its chemical composition - thus fleeing from repellents or moving towards attractants. The key to this system and the first link in the chain is the bacterial chemoreceptor - a membrane protein that binds the effector and transduces the signal down the line. Despite its advantages this system is still rather limited, bacterial chemoreceptors have evolved to sense specific materials that either benefit or harm the cell in some way. The specificity of a receptor is determined by its ligand binding domain. This domain is the focus of project S.Tar.

Fig. 1: A bacterial sense of direction.

S.Tar

The S.Tar platform offers the means to decide what substance will trigger a chemotactic response- controlled chemotaxis. The base of our project and the source of our name is the Tar chemoreceptor, one of four E. coli receptors. By changing Tar’s ligand binding domain to that of other receptors or by mutating it we show that E. coli can be engineered to respond to completely new materials.

As a proof of concept we present two receptors:
1. PctA-Tar , a chimera created by replacing the Tar LBD with that of the Pseudomonas receptor PctA.
2. Histamine-Tar, a receptor created with the help of computational design - using 'Rosetta' bioinformatics software suite to design mutations in the Tar receptor.

Our attempts did not end here, we also present two bold attempts to fuse a ligand binding domain of human origin to Tar in order to induce chemotaxis towards Estrogen hormone and a novel lock and key system which relies on intein proteins . The purpose of these proteins is to disable chemotaxis until a specific substance “unlocks the door” - or in a more scientific way: induces intein splicing and enables chemotaxis.

We have created non-natural chemoreceptors, by modifying Tar’s ligand binding domain.

Fig. 2: Tar chemoreceptor structure. Consists of three main regions: Ligand binding domain (LBD), The HAMP domain and the MH bundle. (2)

References:
1. Bi, S. and Lai, L., 2015. Bacterial chemoreceptors and chemoeffectors. Cellular and Molecular Life Sciences, 72(4), pp.691-708.

2. BI, Shuangyu; LAI, Luhua. Bacterial chemoreceptors and chemoeffectors. Cellular and Molecular Life Sciences, 2015, 72.4: 691-708.‏

3. British Society for Immunology