Team:Utrecht
Project Description
Bacteria need to respond to a wide range of chemical substances which can be harmful or beneficial to them. They use sensory proteins that bind and respond to different chemical substances. These proteins are modular and consist of domains that are highly conserved among different species. Several recombinant proteins have been made in the past showing that their potential to be used as engineered biosensors. Our team will create a living biosensor that can utilize different receptors to detect a broad range of chemicals through an adapted chemotaxis pathway by using bioluminescence. This new diagnostic laboratory tool is re-usable, innovative, inexpensive, flexible, and sustainable compared to current detection systems which often make use of monoclonal antibodies. This new diagnostic laboratory tool is applicable to different areas of research, including concentration-dependent detection of toxins which affect the environment, pathogens, disease states, and pesticides or chemicals affecting agriculture.
Chemotaxis in E. coli is mediated by a highly conserved, specific, and well-studied pathway. It consists of a two-component system, in which a receptor activates the kinase autophosphorylation activity of CheA. CheA subsequently donates its phosphoryl group to the response regulator protein CheY, which translocates to the flagellar motor where it interacts with Flim. This interaction results in a change of the rotational direction of the flagellum. As a consequence, the bacteria reorientates itself in a random way.
Our team will utilize the chemotaxis pathway to create a living sensor that can detect a wide range of chemical substances in a way that is cheaper, easier to use, and faster than the currently used antibody based detection methods. We will do this by expressing a kinase dependent split luciferase (KDSL) assay that measures the phosphorylation state of CheY, in an E. coli UU1250 strain that lacks all wild type chemotaxis receptors but expresses proof-of-principle custom made fusion receptors instead. This assay uses a heterodimerizing luciferase which only dimerizes upon binding of a ligand, by abrogating steric hindrance.
The assay consists of the N-terminal (N-luc2-416) and C-Terminal (C-luc398-550) halves of bacterial luciferase fused to a short linker. This linker consists of CheZ196-214, and CheY53-67 flanked by two 5 amino acid long linker sequences (GGSGG). CheY53-67 contains the D57 residue that is phosphorylated by CheA when the receptor is stimulated due to the binding of a ligand. CheZ196-214 contains the CheY binding site of CheZ. Increased activity of CheA will phosphorylate the D57 residue of CheY53-67. This will allow CheZ196-214 to bind P- CheY53-67. This causes stearic hindrance which prevents the N-luc and C-luc halves of luciferase to reconstitute which abrogates luciferase activity.
We will create two different types of chemotaxis receptors by fusing the ligand binding domains of the cytokinin receptor PcrK, and the epinephrin receptor QseC to the intracellular domain of the E. coli Tar receptor. We opt to use three different fusion points for both receptors based on previously published successful recombinant chemotaxis receptors [1]. We will modify these sensors so they can measure different concentrations of ligand by modifying the methyl accepting residues of the Tar methylation helixes, and expressing different levels of recombinant receptor.
If our proof-of-principle system shows successful concentration-dependent detection of chosen ligands, it will further pave the way for cheap, effective, and modular bacterial-based detection systems.