Expression, purification, and characterization of a carbohydrate‐active enzyme: A research‐inspired methods optimization experiment for the biochemistry laboratory

Abstract

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor.

A Review of Biochemistry Education Research

“…135 Another alternative to authentic research experiences have been described as discovery-based, a term that was applied to a laboratory sequence in which students learned to express a protein using recombinant DNA. 136 Students found this approach "more interesting than traditional experiments because of the lack of a predetermined outcome." Another alternative to an authentic research experience used household materials as a source of DNA and found this approach encouraged creative thinking and improved students' subjectknowledge.…”

Perception of Pre-Service Chemistry Teachers about Detergency: From Macroscopic Observations to Symbolic Theoretical Mental models applied to inactivation of SARS-CoV-2

“…There is a consensus in chemistry and biochemistry that practical activities should be a source of opportunities for students to engage in their own learning [1,[10][11][12][13][14][15]. However, in the context of teaching experimental biochemistry, the "follow the recipe" methodology predominates [16][17][18][19]. Consequently, the active participation of the learner is not encouraged and the methodology cannot be considered effective for teaching Science [20][21][22][23].…”

“…The approach drives the resolution of problems of some relevance to the student [27,28]. Ideally, the proposed problems should favor the construction of knowledge and improve important scientific skills, for example, observation, interpretation, argumentation and discussion [17,29].…”

A Research‐inspired biochemistry laboratory module–combining expression, purification, crystallization, structure‐solving, and characterization of a flavodoxin‐like protein

“…It has been pointed out that an approach with individual laboratory exercises leaves the students lacking an understanding of the big picture . The students often lack the understanding of how the skills they learn in the different laboratory exercises are related, and how they are used together in a real research environment.…”

“…The students often lack the understanding of how the skills they learn in the different laboratory exercises are related, and how they are used together in a real research environment. There are several recent examples of undergraduate biochemistry laboratory courses that have succesfully implemented a more integrated and research‐inspired approach . These courses cover different aspects of the biochemistry education and have different focuses with respect to topics, length, class size, and use of teaching resources.…”