Team:Hangzhou WestLake/Design

Design

1.Generation of plasmids that encodes PCB aptamer. The PCB aptamer will either be engineered at the 5’-UTR before the RBS or within the ORF. This will be carried out by a vendor or generated in the lab.

2. Bacterial transformation. General protocol for bacterial transformation will be carried out using a common E. coli strain (e.g. DH5α). If we decide to use bacterial motility as a readout, bacterial strain lacking CheZ will be used.

3. PCB binding to aptamer. Electromobility shift assay will be employed. Aptamer binding to PCB will cause conformational change of the DNA, which in turn change its mobility. Kd of the aptamer to PCBs can be defined from the gel. Specificity of the aptamer against other structurally similar compounds will also be tested.

4. Efficacy of PCB-regulated gene expression (Fluorescence as a readout). Inoculation of the transformed bacteria with various concentrations of PCB. We expect to see a concentration dependent turn-on (or turn-off) fluorescent signal, depending on the design of the vector. After identifying the maximal turn-on or (turn-off) signal induced by the optimal [PCB], Time-dependent change in expression will be accessed. Bacteria will also be seeded on agar plate with appropriate antibiotics, change in GFP signal can be quantified using microscopy.

5. Recapitulating chemotactic response by using a PCB concentration gradient. Agar plate will be doped with “paths” containing PCBs. Transformed bacteria will be seeded at initial points adjacent to the PCB paths. Depending on the readout, we should expect directional movement of bacteria toward the PCB “paths” (if motility is used as a readout), or specific patterns corresponding to the GFP expression. (if fluorescence is used as a readout).

6. Real sample analysis. Sea/river water spiked with known concentration of PCBs will be doped on agar plate. Similar experiments will be performed as listed in 5.