Dr. Timea Fernandez
College of Arts and Sciences
Department of Chemistry, Physics, and Geology
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The primary objective of this research is to develop an assay – one more feasible for undergraduate institutions than currently available methods – that will enable evaluation of the concomitant binding of, guanidine and tetracycline to the ykkCD bacterial guanidine sensor riboswitch. This noncoding regulatory RNA, in addition to guanidine, also recognizes tetracycline, a translation-inhibiting antibiotic, via a high-affinity aptamer domain. The resulting conformational change by the riboswitch upregulates the expression of genes that are downstream of this regulator, most of which are membrane transporter that expel toxins from bacteria. This phenomenon exemplifies the increasing issue of bacterial antibiotic resistance, by raising the possibility that bacteria uses its existing detoxifying machinery to expel antibiotics thus rendering them ineffective therapeutics.
A binding assay is planned to see if the two ligands, tetracycline and guanidine bind to the riboswitch at roughly the same region, and to investigate the thermodynamics and/or kinetics by which the ligands bind, and to therein see whether or not the binding of the tetracycline antibiotic makes guanidine bind to the aptamer more efficiently. The RNA utilized in this project is from the soil bacterium Bacillus subtilis. Binding assays currently proposed in the literature have called for more cumbersome methods, including the use of radioactively labeled RNAs and the tedious, time-consuming running of sequencing gels, but the goal here is to develop an assay that utilizes the more universally accessible method of isothermal titration calorimetry (ITC) and/or surface plasmon resonance (SPR) to measure the efficiency of guanidine binding to the ykkCD riboswitch while tetracycline is bound. Due to limitations on in-lab experimentation imposed over this summer of 2020 by the ongoing COVID-19 pandemic, a literature review alone is presented here which aids our overall understanding of the project’s current standing and that provides insight as to how we can benefit from applying both or either ITC and/or SPR to make the desired measurements.
When safe and advisable to do so, we intend to physically collect and analyze assay data (going forward with both methods for now) to closely consider the role of tetracycline binding in the life of the regulator and its effect on guanidine binding.
Sullivan, Thomas k., "Elucidating the effect of the antibiotic tetracycline on the regulatory function of the guanidine sensing riboswitch ykkCD" (2020). S.U.R.E Posters. 1.