Difference between revisions of "Team:HZAU-China/Design"

In order to enable bacteria to have the greater targeting ability towards tumor, we improved part OmpA to OmpA-RGD protein to achieve this goal. RGD is a well-studied tumor homing tripeptide that specifically binds to alpha v beta 3 (αvβ3) integrin, which is widely overexpressed on cancer cells and blood vessels during cancer angiogenesis. We intended to display RGD on the surface of bacteria, so as to target the αvβ3 on the surface of tumor cells.

Thus we needed a method to display RGD in the outer membrane. We chose Lpp signal peptide and outer membrane protein A (OmpA) system, which has been extensively used todisplay a diverse group of proteins, including Green Fluorescent Protein(GFP)⁵, Organophosphorus Hydrolase (OPH),single chain Fv fragments (scFv), Cellulomonas fimi exoglucanase Cexand its cellulose-binding domain(CBS_cex)⁶. Therefore, we expressed Lpp-OmpA-RGD fusion protein under the control of Ara promoter in our system. The fusion protein would be anchored in the outer membrane of bacteria and display RGD on the surface. Then RGD would lead the Salmonella to bind with tumor cells which have αvβ3 integrin on their surface⁷.

We decided to use anhydrotetracycline transcriptional regulation system to express GSDMD-N275 protein. It is one of the commonest methods to express toxin protein because of its low expression noise, high response speed and the good linear relation between the inducer and the expression of product. Anhydrotetracycline transcriptional regulation system is a one-component signal transduction system that regulates the expression of the tetracycline resistance determinant encoded by tetA, the anhydrotetracycline efflux pumping in Escherichia coli⁸. In this system, promoter P_tet expresses the repression protein TetR. With the presence of ATc (anhydrotetracycline), TetR will integrate ATc and. This induces a conformational change in TetR and its dissociation from the operon of P_tet and abolishes the repression⁹.

We decided to use this system to regulate the expression ofour GSDMD-N275 protein, which controlled by P_tet promoter.

However, we worried that the leakage of GSDMD-N275 will induce cytotoxicity. So we replaced different strength of RBS to choose the suitable RBS to control the leakage of GSDMD-N275. Owing to testing the linear stability of ATc induction and to transfer the expression of GSDMD-N275 to the concentration of ATc, we also constructed the TetR::EGFP (enhanced green fluorescent) fusion protein in E.coli (Fig.5) as an essay suggested¹⁰. And we induced Salmonella with gradients of ATc and measured the fluorescence accordingly. More details to our experimental method has been listed in our notebook and the ATc induction model. Finally, we constructed the EGFP::GSDMD-N275 expressed by the anhydrotetracycline transcriptional regulation system to proof our final circuit.

The Intracellular Specific Induction

Once delivered into the host cell, Gram-negative bacteria Salmonella always express its virulence genes to alter host cell functions with the advantage of the pathogen. These genes are in Salmonella Pathogenicity Island and enterobactin gene cluster^1,11. Owing to the feature of expressing intracellularly, we used promoters of these genes to express GSDMD-N275 after its entrance to tumor cells. To achieve this goal, we constructed P_sifA::GSDMD-N275 and transferred it via electroporation (see details in our Methods). The figure below shows their functions (Fig 6.). Promoters in the circuits won't work outside cells but only express GSDMD-N275 after the entry to tumor cells.