In class, while the overall format was lecture-based, the students actively participated by examining figures and gels from the text. The lecture outline was available before class on the course website so students could familiarize themselves with the upcoming lecture. This meant more class time was available to analyze experimental data and to discuss articles, which were also available on-line. Techniques used in experiments were described and tied to techniques being used in the laboratory section of “Molecular Biology.” Approaches to research were also discussed in the context of the 50th anniversary of the discovery of the structure of DNA and the difference between discovery-based and hypothesis-driven research. Students were encouraged to participate and comments made by the group were written on the board for later reference. Some of these same issues appeared on quizzes or exams (see assessment section).
The students in this “Molecular Biology” course successfully cloned two genes from the bacterial chromosome, the hns gene (described below) and a gene of unknown function, yjjQ. The hns gene encodes a DNA binding protein of ∼15,391 Da that acts pleiotropically in Escherichia coli as a global regulator of gene expression. The students cloned PCR-generated DNA carrying the chromosomal hns gene into a protein expression vector, verified the presence of the insert, and then used SDS-PAGE to visualize the protein. This cloning project, which started fairly early in the semester, gave the students a sense of ownership and involvement in the “Molecular Biology” laboratory section.
Qiagen generously donated the protein expression vector pQE31, which is designed both for inducible gene expression and the addition of a histidine (His6) tag on the N terminus of the expressed protein. Three vectors are available, one of which corresponds to the reading frame of the protein of interest. The sequence of the H-NS coding region was in a different reading frame than pQE31, and its gene needed to be adapted before cloning into pQE31. Students were given the assignment to design primers containing restriction enzyme sites and the proper number of nucleotides so that the amplified hns gene could be inserted into the vector with the hns sequence in frame with the his codons. This was a challenging assignment, which required that the students understand the process of amplification and how new restriction sites can be incorporated into a genetic sequence. They had to grapple with reading frames so that the His6 tag could be added to the H-NS protein. Suddenly reading frames, with which they have all been familiar, took on new meaning and became more than just a homework assignment. The students needed to figure this out in order to proceed in cloning the hns gene. Not only did they want to clone the gene, but also they wanted a properly translated protein.
The students were given microgram amounts of PCR products to facilitate cloning or set up the PCR themselves. The key point was they needed adequate amounts of PCR product to be successful in cloning. Qiagen kits were used for PCR purification, gel extraction, and minipreps (see below). The students digested the purified PCR product and the plasmid vector with restriction enzymes, purified the fragments by gel electrophoresis, and carried out a ligation reaction based on their estimates of vector to insert ratios. The next laboratory period, they transformed XL-1 Blue (Stratagene, La Jolla, CA), which is an excellent cloning strain. Nearly all of the students had transformants the next day, and in the following laboratory period, they isolated plasmid DNA from their transformants. To determine if they had actually cloned the hns gene, they digested their plasmid DNA with restriction enzymes and looked for the presence of an insert on an agarose gel. Three groups out of seven had plasmids containing the hns insert. E. coli strain BL21 (Pharmacia), which is useful for protein expression, was then transformed with the plasmids carrying the hns gene. Cultures of BL21 were grown with or without isopropyl β-D-thiogalactoside (IPTG) to induce expression of the gene. The students lysed the cells by boiling in SDS sample buffer and then carried out SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R, and the H-NS protein could be clearly seen in the total protein extract in cultures induced with IPTG. The students succeeded in cloning a gene from the chromosome and visualized its protein product under conditions that they controlled.
Earlier in the laboratory sequence, the students learned how to isolate plasmid DNA, digest DNA, and transform cells. Now they used these methods with their own clones to determine whether or not their plasmids contained inserts. The repetition of these methods was beneficial in that they could see the utility of these techniques in cloning a gene. There was interest to see who had the insert as well. At each step of the cloning procedures, students realized that, for example, ligations may or may not work and transformation with the ligation mix may or may not result in transformants. They had to figure out what affects these procedures and try to come up with optimal conditions. Understanding induction became crucial as well; no longer was regulation of the lac operon an abstraction because the plasmid they were using had a phage T5 promoter regulated by two lac operator sequences.
The key strategy in this course was to have the students work independently without daily procedures being provided to them. There is a theme to the course each semester, with protein purification as the theme of the semester being discussed, and a suggested global methodology, such as using GST fusion purification techniques. To cut down on the reagents/resources that the instructor needed to provide, the students were given a defined range of methods. They formulated a hypothesis about an undetermined outcome and then designed and planned the experiments that would address that hypothesis. The students chose techniques within the loosely defined theme, implemented the experiments, and then reported to the instructor as an advisor. The students did all the background research themselves and used other students as resources. Then they chose the variables with which to experiment when trying to optimize methodology. There were weekly laboratory meetings where students received peer feedback on their experiments, just as in a graduate laboratory. They were required to maintain a detailed notebook and protocols book. The students approached the project with the expectation that a thesis in the form of a manuscript would be the final outcome.
As described above, students used basic biochemical and molecular biological techniques to purify a protein using the GST fusion protein purification system and followed suggested protocols provided by the manufacturer, Amersham Pharmacia Biotech, Inc. They were to purify and solubilize a protein to the extent that others could then use the pure protein in biological assays. They were given a choice of two proteins to work with: BglJ, a protein involved in anti-silencing in Eschericia coli, or FHOS, a novel formin protein with a potential role in signal transduction. These proteins were chosen because they are components of ongoing research in faculty laboratories here at Simmons. The use of real research problems enhanced the students' feelings of investment in the final outcome of their endeavors. Students were provided with background literature on the methodology and on the target proteins, as well as the manual that accompanies purchase of the materials from Pharmacia, which were graciously provided by the company . At the start of the semester, the students were guided through the Pharmacia manual and they were given instruction to initiate the experiments. After the first 2 weeks, they were responsible for planning each subsequent experiment based on analysis of the data from the previous ones.
Initially, students needed to check the DNA that they were given to confirm that the plasmids did indeed contain the DNA sequences corresponding to bglJ or FHOS, respectively, which encoded the two proteins of interest. They isolated plasmid DNA using Qiagen kit technology and then they used restriction analysis to verify the presence of an insert of the expected size. They transformed the plasmid DNA containing the sequences encoding the GST protein fusions into BL21 cells. Then they began their quest for purification. The two proteins chosen for purification have markedly different structures and functions. This demonstrated key principles of how protein structure can affect solubility and whether or not a protein can be easily purified.
Upon verification of the cloned GST constructs, the students began their purification procedures, asking the relevant questions in order to determine the best strategies for experimentation. For example, students asked:
After transforming BL21 cells with the GST-BglJ plasmid and inducing expression of the fusion protein with IPTG at 37 °C, was a protein at the expected size observed on a Coomassie-stained SDS gel?
Was there expression of the GST protein alone as a control?
What was the length of induction time?
What growth temperature worked best for protein expression?
Did temperature affect potential solubility?
Each of these questions gave students opportunities to troubleshoot and enhance the protocols in order to optimize results. At this stage, the instructor was primarily available for consultation and guidance, but the ultimate decisions about what variables to manipulate were determined by the students. This gave the students a sense of ownership of the outcome and allowed them to feel fully involved in their research. As the students progressed, each group of students was ultimately pursuing different hypotheses and no two groups were performing the same experiment. This provided an environment similar to a real research laboratory and fostered communication between laboratory groups. Students used each other as sounding boards and colleagues, often sharing results that could help other groups in planning the next steps.
Students repeated the expression assays many times and manipulated some new aspect of the protocol to both enhance the amount of fusion protein expressed and to increase solubility. The goal was to produce the protein in sufficient quantities so that the GST tag could be cleaved and the purified protein used in enzymatic assays. They became experts with SDS-PAGE gels and Coomassie staining. They manipulated culture temperatures, culture media, protein concentrations, and induction times with each subsequent experiment. At the conclusion of this course, students were able to show successful expression of both GST-BglJ and GST-FHOS in BL21 cells. They subsequently demonstrated increased solubility of both fusion proteins upon manipulation of the induction time and culture temperature. They successfully performed a Western blot using a GST antibody probe and went on to cleave the GST-BglJ fusion using the thrombin enzyme to produce purified BglJ protein.
Active participation in the decision-making process contributed greatly to their comprehension of the difficulties facing a researcher at the bench. In “Advanced Biochemistry,” each group of students working on a particular project prepares a manuscript of their results. Like any laboratory setting, some projects are more feasible than others and some students make more progress. Because each group of students was responsible for a particular project, their manuscript reflected their progress. If the manuscript is of publishable quality, students were encouraged to submit it to a journal publishing student research with the assistance of the faculty members. Seeing laboratory work go from the bench to a manuscript is a powerful experience for all the students whether they are able to publish or not. Students felt a tremendous sense of accomplishment at the end of the semester, and one group went on to publish their results in the Journal of Undergraduate Chemistry Research .