Difference between revisions of "Team:Technion Israel/Proof"

PctA-Tar chimera Introduction

One of S.Tars sub projects focused on altering and changing the LBD of the Tar chemoreceptor in order to design new hybrid chimeras. These changes were made by replacing the LBD of the original Tar chemoreceptor with a new one, from a different source, while keeping the signaling region of Tar untouched. As a proof of concept for the newly designed Tar chimeras and the S.Tar project, we focused on testing the PctA-Tar hybrid.

Fig. 1: Scheme of native Tar chemoreceptor, native PctA receptor and PctA-Tar chimera. Adapted from (1)

PctA is a chemoreceptor found in the Pseudomonas Aeruginosa bacterium, it mediates chemotaxis towards amino acids and away from organic compounds. It can sense all amino acids except for Aspartate (1).
To construct this chimera, the LBD sequence of the PctA was obtained from the Pseudomonas genome database, while the signaling region of Tar was obtained from the iGEM parts catalog (K777000). Using these two sequences, we built a Biobrick device (K1992007) which was then transformed to bacteria that lacks chemoreceptors - UU1250 , to be extensively tested. It is important to note that this chimera has been constructed before in the literature (1).

Finally, demonstrated below is a working concept of the FlashLab project - a chip that serves as a detection tool based on the chemotaxis system of E. coli bacteria - by using a commercial ibidi chip filled with a suspension of bacteria expressing the chimera and chromoprotein (J23100 + K1357009). A solution of Tetrachloroethylene in concentration of 10^-3M, the repellent, was added to the chip and the displacement of the bacteria was monitored and recorded.

Fig. 8: A steps scheme of the FlashLab concept: Add bacteria expressing the chemoreceptor of your choice and a chromo protein to a fluidic chip . Add the sample in question to said chip. If the sample contains the substance that is recognized by the chemoreceptor, a displacement of the bacteria will become visible. If not, then the no displacement will be seen.

With the supporting evidence of the results presented above, it can be concluded that both concepts have been proved and work under real life conditions and might lead to the detection of various substances in the near future.

Histamine-Tar Introduction

The base of the S.Tar project is the Tar chemoreceptor, one of four E. coli receptors. Our goal is to create a engineered bacteria which has chemotaxis receptors sensetive to materials outside it’s existing receptor base.By changing Tar’s ligand binding domain (LBD) to that of other receptors from various sources or by mutating it we show that E. coli can be engineered to respond to completely new materials.

Fig. 1: Sequencing Results. Query describes the native Tar LBD sequence and Sbjct describes the design mutations sequence. Each mutation regin marked with another color (blue and red).