Preface

Preface

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 21^st century 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.

A biochemistry laboratory course is required for chemistry majors, biochemistry majors and may be chosen as an elective by pre-health professionals or students focusing on the molecular aspects of biology. This laboratory manual takes advantage of the fact that a biochemistry lecture course is pre- or co-requisite to taking a biochemistry lab course. As a result students are expected to be familiar with the general principles behind each experiment from a lecture course. The organization of each chapter is as follows

– Review of principles: Each chapter begins with a to-the-point review of basic principles (what is a nucleic acid, what does a polymerase do and how it works etc.). In the authors’ experience, limiting background in a lab manual to information that is pertinent to performing the laboratory experiment is a good strategy at the undergraduate level. This approach directs students’ attention to putting the theory into practice as opposed to learning the theory in lab, which can be done better in the lecture course. In other words, we find that when we teach lab with a clear focus on improving students’ experimental skills we achieve a higher content retention.

– Reagent needs: A complete list of materials and equipment needed is listed for six student teams. Two-to-three students per team works well to maximize peer interaction while still making sure that each student has a chance to intellectually contribute to the assignments. The course was typically taught by a trained biochemist and a teaching assistant at the authors’ institution, but one instructor is sufficient for a group of no more than 16 students.

– Protocols: A detailed protocol is given for each experiment, including recommended timeline to complete experiments within three hours, which typically is the time allocated for a biochemistry laboratory session.

– Notes to the instructor: This section provides information on how to (a) alter the protocol to accommodate different instrumental setups; (b) arrange the experiments to fit within the three hours lab time that is commonly used at most institutions; and (c) how to utilize the waiting time that is inevitable when performing biochemistry experiments.

– Problem sets: Problem sets are grouped into three categories:

– Pre-lab questions are designed to focus students’ attention to the most important points in the experiment. In the authors’ experience, students perform much better during lab if they are asked to answer simple questions about the experiments ahead of time. Therefore, it is recommended that students complete pre-lab assignment before coming to lab.

– Lab report checklists contain questions that guide students through data processing and analysis.

– Worksheets contain problems that are designed to help students think more closely about each experiment. These questions are of increasing difficulty. The instructor may assign all the questions or pick ones that best match the skill level of the class. They work well as problem sets during lab to fill the waiting time that is notorious for biochemistry experiments.