Difference between revisions of "Team:Utrecht/Results"

Explore the possibility of using the ligand binding domains from different transmembrane receptors.

Approach

In its final form DeTaXion will use customized receptors that can detect different compounds. These receptors will be designed from a variety of existing transmembrane receptors (from the chemotaxis and other pathways), and further engineered with the help of prediction software such as Haddock, to provide the best possible binding affinity between receptor and ligand.

To provide a proof of concept within the scope of this project, we tested the possibility of swapping ligand binding domains.

Results

We created a hybrid receptor consisting of the intracellular domains of the bacterial copper sensing protein CusS, fused to the ligand binding domain (LBD) of the Tar receptor (Figure 1). The Tar receptor is one of the main receptors of the chemotaxis pathway, and senses Aspartate (Bi et al., 2013). CusS is part of a two-component signaling pathway and senses copper and silver ions. In the presence of copper, CusS phosphorylates the response regulator CusR, which in turn interacts with a copper responsive promoter element, activating gene expression (Munson, Lam, Outten, & O’Halloran, 2000). We chose this configuration because it would allow us to test the possibility of generating hybrid receptors using a simple transcriptional readout, without having to be dependent on Aim 2 (generation of a functional BRET pair).

To test the functionality of our receptor, we generated a new BioBrick (BBa_K2736105) consisting of a copper sensitive promoter followed by the coding sequence of the red fluorescent protein (RFP, from BBa_K516032) (Figure 2). By measuring the intensity of RFP emission in a photospectrometer or by fluorescence microscopy, we can determine both whether the receptor functions, as well as to what degree it transduces a signal.

We transformed E. coli strain DH5α with plasmids encoding the RFP reporter, as well as our hybrid receptor. As a control, we transformed E. coli with the reporter alone. We grew both strains overnight in LB, which contains Aspartate and hence should result in RFP expression when our hybrid receptor is present. After overnight culture, we assessed the presence or absence of RFP by fluorescence microscopy. We observed bright RFP expression in bacteria transformed with both receptor and reporter encoding plasmids, but only very low levels of RFP in bacteria transformed with the reporter construct alone (Figure 3).

To further test the responsiveness of the Hybrid receptor to Aspartate, we attempted to grow our E. coli strain in a minimal medium lacking Aspartate (M9 + 0.4% glucose), which should not induce expression of RFP. However, we were unsuccessful at culturing bacteria in this medium. As an alternative, we grew bacteria overnight in LB (causing RFP expression), and then transferred them to M9 + 0.4% glucose for 8 hours to allow part of the RFP to degrade. We then split the bacteria over two tubes, and added 250 uM Aspartate to one of these samples. After 4 hours, we quantified the levels of RFP expression using a spectrophotometer. Even after 8 hours in M9, levels of RFP were still very high. Nevertheless, we observed slightly elevated levels of RFP in the bacterial culture incubated with Aspartate, compared to untreated control (Figure 4). While this indicates that our receptor is responsive to Aspartate, this result will need to be strengthened in future experiments.

Conclusion

The expression of RFP in bacteria transformed with hybrid receptor and RFP reporter, but not in bacteria transformed with reporter alone, demonstrates the functionality of our copper-sensitive RFP reporter (submitted part BBa_K2736105). Is also shows that its expression is dependent on the presence of the hybrid Tar/CusS receptor. Presumably the expression is due to the presence of Aspartate in LB medium. The slight increase in RFP expression upon addition of Aspartate after incubation in M9 medium (lacking Aspartate) for 8 hours further indicates that our receptor is indeed responsive to Aspartate. Together, these results demonstrate the feasibility of using alternative ligand binding domains to broaden the range of targets detectable by DeTaXion. Key future experiments include further investigation of the Aspartate responsiveness of the hybrid receptor, and the generation of receptors carrying ligand binding domains engineered to bind pharmaceutical compounds.

Aim 3: Adjusting the dynamic range of the chemotaxis system to enable detection of a broad range of contaminant concentrations.

Approach

To facilitate the measurement of multiple ranges of concentrations, we adjusted the sensitivity of the receptor. This approach is based on the methylation state of the Tar receptor, which influences its sensitivity to ligands. In the unmethylated state, the receptor efficiently transduces the ligand binding signal to the downstream pathway (see project design). Upon receptor methylation, the conformational change required to transduce the signal, is energetically less favorable. Therefore, when the same amount of ligand is present, the level of signal transduction is lower than in the unmethylated receptor state. The Tar receptor has four acidic residues that can be methylated (Q295, E302, Q309, E491). The methylation of glutamine (Q) or glutamic acid (E) residues can be mimicked by mutating these residues to alanine (A) (Krembel et al. 2015).

Results and discussion

Our computational modeling indicated that the mutation of the methylation sites should indeed result in altered sensitivity of the receptor. We therefore chose to generate three different methylation state Tar variants:

1. The wild type state, the unmethylated Tar receptor (QEQE, most sensitive);
2. A Tar receptor with one mimicked methylation site (QEAE, average sensitivity);
3. A Tar receptor with two mimicked methylation sites (QEAA, least sensitive).

We received the wild-type Tar receptor from a collaboration with iGEM team Groningen. We next used site-directed mutagenesis to introduce the Q309A and E491A mutations, resulting in BioBrick parts BBa_K2736108 and BBa_K2736106 respectively. Sequencing confirmed the successful generation of these components. However, because the BRET assay was not yet functional, we were unable to test the relative activity of these Tar receptors in the time frame of the competition.