The purpose of this laboratory manual is to introduce undergraduate students to techniques used in biochemistry and molecular biology laboratories and ensure that they master the lab skills necessary to be competitive in the job market. We present a collection of fifteen experiments that teach students sterile techniques, accurate pipetting, centrifuge usage, PCR, DNA purification, protein expression and purification, HPLC, enzyme kinetics, equilibrium binding assays and introduction to bioinformatics. The emergence of bioinformatics is one of the biggest change that happened to biochemistry in the last two decades. The availability of genome sequences increased exponentially, online data banks and free programs are now available to make sense of these data. As a result we can learn about a biomolecule before ever lifting a pipette in the lab. These resources are invaluable to today’s biochemists when they set up a working hypothesis. We expect a continued increase in the availability of data mining programs that help interpret the tremendous amount of genome sequence, structure, microarray etc. data thus preparing 21stcentury biochemists to use these programs is a must. To our knowledge, this is the only manual that includes several chapters on the latest advancements in bioinformatics: how to access genome databank, perform sequence alignments, design primers, to predict secondary and tertiary structure and to use protein visualization tools.
The unique feature of this laboratory manual is a hypothesis-driven real-life research project. In this project, students study how a noncoding RNA molecule that plays an important role in bacterial antibiotic resistance recognizes its target antibiotic. By including a research project in the undergraduate lab, students learn how real-life research works: first they set up a hypothesis then design experiments to test the hypothesis and finally evaluate the hypothesis using a functional study. With this experience students get as close to doing a research project as possible within the framework of an undergraduate laboratory. During the nine-weeks project incorporated into this laboratory manual students perform sequence alignment to determine evolutionary conserved elements in the noncoding RNA, they design primers to make mutants using site-directed mutagenesis then synthesize and purify the noncoding RNA mutants. Finally, they test the ability of the mutants to recognize a target antibiotic using a fluorescence-based binding assay. A big challenge in teaching upper level labs is the very different background and experience level students come to the course with. Some students have no biochemistry lab experience while others have done research as an undergraduate for years. Since students test their own mutant design even the most experienced students remain engaged with the process while the less experienced ones get their first taste of biochemistry research. The design of the mini project is flexible: experiments may be done in a different order or on a different target. At the authors institution the nine-week long research project was taught in the order listed in the manual: mutant design, synthesis and functional study. Alternatively, the order may be reversed: students start with analyzing a previously made noncoding RNA mutant and based on their findings they design a better mutant. A collection of ykkCD RNA mutants is available from the authors’ upon request. As written, the mini project is designed to understand how the ykkCD noncoding RNA recognizes its target antibiotic tetracycline, but the project with minimal modifications can be used to examine any biologically important noncoding RNA, such as a ribozyme or regulator.