Developing a High-Throughput LacZ Reporter Gene Assay to Evaluate Activation of the xpt-pbuX Guanine Riboswitch by Analogue Ligands

Riboswitches are special structures of RNA typically found in bacteria. They respond to the presence of certain molecules, called ligands, within the bacterial cells to regulate expression of certain genes. Genes expression is the process by which information encoded in a segment of an organism’s genetic material is used to produce functional molecules utilized by that organism. Riboswitches are currently being researched as a possible new class of antibiotic targets amid the growing public health threat of antibiotic resistance. In order to compare the effects of guanine (the natural ligand of the riboswitch), guanosine (a biological molecule similar to guanine) and SK4 (a molecule synthesized by previous Bard College seniors based on the structure of guanosine) on the xpt-pbuX riboswitch, I used a reporter gene assay in a model organism: a harmless type of bacteria called Bacillus subtilis. The structure of the xpt-pbuX riboswitch allows it to interact with ligands of shape similar to guanine. For the reporter gene assay, a gene (called LacZ) that encodes the enzyme beta-galactosidase is specially inserted into the bacterial genome next to the riboswitch. The production of this enzyme is measure by adding a chemical called ONPG to the bacterial cultures. The enzyme reacts with ONPG to produce a yellow color that can be measured. The less intense the yellow color, the less active enzyme has been produced by the bacteria. Thus, expression of the LacZ gene “reports” back on the activation of the riboswitch. If incubating bacterial cultures with a given ligand results in less production of active enzyme as compared to incubation without the ligand, this is presumed to mean that the ligand has activated the riboswitch. In this project I worked to develop an alternative method for the traditional beta-galactosidase assay, minimizing the quantities of chemicals and bacterial cultures required. Instead of incubating bacterial cultures in vials with the study ligands and using cuvettes of bacterial cultures for the enzyme assay, I incubated very small volumes of bacterial cultures in the wells of 96-well microplates and then performed the assay in 96-well microplates. The intensity of yellow color in these bacterial cultures can be directly measured in the microplates using a plate-reader. I also implemented a kinetic assay in which the changing intensity of yellow color is monitored over time instead of waiting for yellow color to develop and then measuring it, with the hope of minimizing timing error in the assay