Site-Directed Mutagenesis Study of an Antibiotic-Sensing Noncoding RNA Integrated into a One-Semester Project-Based Biochemistry Lab Course

A laboratory sequence is described that is suitable for upper-level biochemistry or molecular biology laboratories that combines project-based and traditional laboratory experiments. In the project-based sequence, the individual laboratory experiments are thematically linked and aim to show how a bacterial antibiotic sensing noncoding RNA (the ykkCD riboswitch) recognizes its target antibiotic, tetracycline. The novelty of the curriculum lies in its extensive coverage of bioinformatics as well as its focus on synthesis, purification, and functional studies of a biologically active RNA. Further advantage of the curriculum is that the model system, the ykkCD riboswitch, regulates expression of a gene that is involved in bacterial antibiotic resistance. Hence, the project makes this important issue part of the undergraduate biochemistry curriculum. The curriculum is available in Open Access format as part of a biochemistry laboratory manual by Gerczei and Pattison. This manuscript summarizes the curriculum and provides suggestions to the instructor regarding its implementation.