A research project-based and self-determined teaching system of molecular biology techniques for undergraduates

Authors

  • Shuping Zhang

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    1. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
    • Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
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    • Tel.: 86-10-62773628; Fax.: 86-10-62794907


  • This work is supported by: National Science Fund of China for Fostering Talents in Basic Science (grant no. J0630647) and 985 General Project of Tsinghua University for Undergraduate Teaching.

Abstract

Molecular biology techniques play a very important role in understanding the biological activity. Students who major in biology should know not only how to perform experiments, but also the reasons for performing them. Having the concept of conducting research by integrating various techniques is especially important. This paper introduces a research project-based and self-determined teaching system of molecular biology techniques for undergraduates. Its aim is to create an environment mimicking real research programs and to help students build up confidence in their research skills. The students are allowed to explore a set of commonly used molecular biology techniques to solve some fundamental problems about genes on their own. They find a gene of interest, write a mini-proposal, and give an oral presentation. This course provides students a foundation before entering the research laboratory and allows them to adapt easily to real research programs.

Our understanding of living things at the molecular level has undergone rapid development in the recent years. Molecular biology techniques are considered to be powerful tools in making breakthroughs in understanding the biological mechanism. Therefore, students majoring in biology must acquire extensive experience in working with biomolecules in the laboratory. Besides this, a wide range of related professional careers, including medicine, public health, the pharmaceutical industry, scientific publishing, and even biotech business, will also benefit from good research practices. As a result, a systematic teaching system of biological experimental techniques has been in demand for those college students who study modern biology. It is a good teaching philosophy that students should be inspired to pursue knowledge itself and contract the habit of raising questions, and even to challenge the existing evidence, hypotheses, and concepts [1, 2]. It is believed that students can gain much more knowledge and ability by active learning than passive learning.

Molecular biology techniques are developing at an accelerating pace. Undoubtedly, the teaching laboratory has become a prominent and essential fixture in the training of undergraduate students for careers related to the life sciences [3]. Obviously, it is impossible for undergraduates to explore all of the techniques in one semester and on a limited budget. In fact, it is not necessary to do so. Instructors should know how to tutor and organize the experiments for the greatest benefit to the students. This greatly helps students master the skills and principles, and even develop their interest in biological science. There are many papers demonstrating that integrated and research project-based courses are very beneficial for students' understanding [3–5]. However, courses that both are research project-based and allow student self-determination have been less explored. This paper introduces a research project-based and self-determined teaching system of molecular biology techniques for undergraduates, and emphasizes the importance of a systematic teaching system and self-determination. The results demonstrated that this kind of course develops students' creativity and inspires their enthusiasm for scientific learning.

COURSE BACKGROUND AND PROJECT SELECTION

Tsinghua University presently offers a one-semester course of basic Molecular Biology Laboratory for senior students or some sophomores in the Department of Biological Sciences and Biotechnology. This course is guided step by step by instructors to expose undergraduates to a set of standard and basic molecular biology research techniques, including DNA extraction, electrophoresis, enzyme digestion, polymerase chain reaction (PCR),1 DNA recombination, RNA manipulation, and molecular hybridization. Because the coursework is derived from an actual research project, all experiments are interrelated, although each can stand alone as an independent experiment. At the end of this course, students are required to synthetically use these techniques or some new techniques to design an integrated experiment, but it is not put into practice in the same term. On the basis of the first course, some students can choose an advanced course in the summer term, which is organized to mimic a real research program. This course is largely self-determined and challenging. Students work in small groups made up of 3–4 students. They choose projects, write proposals, design procedures, and carry out all the experiments by themselves while instructors only give them some suggestions. They must read other papers and discuss all the details with their group members or other classmates. As soon as the students elect this course, they are asked to improve and rewrite an integrated experiment they designed in the basic course to a small proposal, and resubmit it 2–3 weeks ahead of the time they will start their laboratory work so that the instructors will have time to review all the proposals and order the necessary reagents.

The projects are required to mainly focus on molecular biology research. The students can choose their projects either based on the experiments they designed in the basic course or combined with some faculty projects in the research laboratories. They are allowed not only to use techniques learned in the basic course (as mentioned earlier), but also to try some new techniques that they did not learn; these techniques must be capable of completion in the summer term.

Considering that the ideas and skills of the students are not very mature at this stage, the instructors should provide some suggestions related to effective time management, organizing experiments, and creative problem solving, to avoid some obvious unrealistic and unreasonable projects and to prevent problems. The proposal should include the introduction of the research background, purpose, scheme, materials and methods, and predicted results (even including the budget). Finally, each group is required to give an oral presentation, a paper report, and a summary. This course is carried out in the summer term and requires working every day in the laboratory for 3–4 weeks. Based on the space and budget, 30–35 students are enrolled each term. The instructor group includes at least one teacher, one laboratory assistant, and two teaching assistants. The teaching assistants are PhD students from research laboratories and have some experience in molecular biology.

MAJOR EQUIPMENT AND REAGENTS REQUIRED

The following major pieces of equipment are requisite: autoclave, culture incubator and shaker, microcentrifuge, UV spectrophotometer, thermal cycler, UV imaging system, water bath, horizontal and vertical mini gel electrophoresis apparatus, and electrophoresis power supply.

The kits and enzymes for RNA isolation, reverse transcription PCR (RT-PCR), normal PCR, mutagenesis, plasmid preparation, DNA purification, digestion, and ligation need to be ordered in advance. Reagents for protein induction, gel electrophoresis, protein staining and destaining, and making different buffers are prerequisite. Some vectors are provided for students to choose from and several bacterial strains are needed as well.

OBJECTIVES

One of the purposes of the university is to provide students with excellent training. Education reform calls for the continued improvement of teaching standards and pedagogical research. We care about how well our students are prepared for subsequent study and work. For students majoring in biology-related disciplines, laboratory practice is especially essential. Therefore, the following objectives are emphasized in this course:

  • 1)To develop students' understanding of the concepts and principles learned from molecular biology and their significance in life science.
  • 2)To show students how to initiate typical molecular biology projects.
  • 3)To show students how to write a proposal, conduct a research project, and analyze data.
  • 4)To inspire the students' interest in research and get students more involved in the experiments.
  • 5)To show students how to systematically solve some basic problems of molecular biology by scientifically applying various techniques.
  • 6)To encourage teamwork, mimicking that in a large scientific laboratory.
  • 7)To encourage students to extend their thinking beyond the provided manual and draw inferences about other cases from one instance.
  • 8)To encourage students to ask questions during oral presentations such as scientific lectures.
  • 9)To emphasize hard work, responsibility, persistence, discussion, and persuasion.
  • 10)To show students how to present scientific thoughts and data both orally and in writing.

MEANS OF EVALUATION

As mentioned earlier, the main purpose of this course is to provide students with integrated training in a simulated research environment. The main focus is how much the students gain. The evaluation does not consider the final results as the only standard for evaluating the students' work, because not all of the projects chosen by students will have been pretested, which means that not all groups will be able to reach their final goals as described in the proposals. This is an embodiment of a student-centric approach. Students are graded as a group. The final scores are calculated according to the following components:

  • 1)Proposal (15%): The proposal is evaluated according to how well the students understood the background, how many papers they reviewed, what their detailed protocols and plans were, and what materials and methods they planned to use.
  • 2)Attitude (10%): The students are required to fully work in the laboratory during the course. The effort, working time, ability to solve problems, responsibility, and collaboration with classmates are evaluated. This encourages students to effectively manage time, cooperate, and produce their results as a team effort.
  • 3)Presentation (10%): Articulate expression and proficiency are imperative for good researchers, so the students are required to clearly present the background, objectives, progress, results, problems, and solutions.
  • 4)Results and notebook (20%): The results are evaluated step by step, such as the results of bioinformatics analysis, RNA isolation, primer design, target fragment amplification (some group only wanted to get some deletion), gene cloning and identification, protein expression and purification, and so on. Evaluation of the results depends on the differences between the projects. All data must be labeled as those published in formal journals, and maps should be drawn of all constructed plasmids to show the backbone, antibiotic resistance gene, and enzyme sites for inserting the target gene. Taking good notes is also a part of the whole project. Faithful recording of every detail is very important for a good researcher, whatever the results may be, whether as expected or not.
  • 5)Final report (40%): This is the largest part of the evaluation of the students' work. Logical organization of the whole report, clear interpretation of the results, reasonable discussion of the problems, and the length are the main criteria assessed.
  • 6)Summary (5%): The students are required to briefly summarize their experience, achievement, and performance. Suggestions for improving the course are especially welcome.

POINTS AND CONCEPTS MAINLY ADDRESSED

This course not only gives students a taste of the research process, but also helps them to deeply understand some of the essential points and concepts of molecular biology. In most basic laboratory courses, the instructor does most of the design work, so the students do not gain much experience in planning and organizing the research projects. This course gives the students an opportunity to have a systematic practice and teaches them to apply the following points and concepts during their experiments.

  • 1)Finding genes: Students must learn to use biology websites, especially the NCBI GenBank database, to find and download the sequence of the target gene.
  • 2)Bioinformatics analysis: Students learn to analyze the gene structure, chromosome localization, homologous alignment, conserved protein domains, and phylogenetic tree by using various websites and software, such as http://www.ncbi.nlm.nih.gov/BLAST/, Bioedit, and DNA Star.
  • 3)Enzyme analysis: Students learn to analyze the enzyme sites in both the target DNA and vector sequences to find suitable sites for cloning and understand the meaning of unique enzyme sites.
  • 4)Primer design: Students learn to design primers by considering the length, melting temperature (Tm), GC content, duplex formation, and hairpin formation, and understand the significance of adding enzymes sites and protection bases at the 5′ end for cloning.
  • 5)Open reading frame (ORF) and fusion protein: Students should learn the concept of ORF and the meaning of the coding sequence for a gene; they should learn to express fusion protein by considering protein ORFs as well as start and stop codons.
  • 6)Structure of the vector: Students should learn to choose vectors and understand the essential components that define a vector. Because different vectors have different usages, students should know what kind of vectors are used for expressing protein in prokaryotic cells and eukaryotic cells.
  • 7)PCR condition settings: Students should learn to set PCR conditions by considering the Tm of primers and the concentration of other components. They should learn the characteristics of different DNA polymerases, for example, what kind of DNA polymerase has proofreading activity, what kind of DNA polymerase does not have proofreading activity, and with what kind of ends the PCR fragment can be used to perform TA-cloning.
  • 8)Point mutation or mutagenesis: Students should learn the meaning of mutagenesis, because mutagenesis is an important concept in biology classes. There are many commercial kits that can be used to achieve point mutation and students should understand that splice overlap extension (SOE) PCR also is an alternative way to perform point mutation or deletion.
  • 9)Tissue expression pattern: Students learn to interpret the results of tissue expression, because tissue expression patterns can provide significant information about where a gene may function. If a novel gene is chosen to be characterized, the tissue expression pattern can be determined by semiquantitative RT-PCR.
  • 10)Safety considerations: Students should gain a consciousness of safety, both in relation to the students themselves and the surroundings, when handling hazardous materials, such as ethidium bromide (EB) and UV light.

ORAL PRESENTATION

Each group is required to give two oral presentations to the whole class: one in the midterm and another at the end of the term. In the midterm, the students mainly report their research background, procedures, progress, and problems. At the end of the term, the students report their results, analysis, discussion, and experience. The students are encouraged to ask questions and make suggestions during or after the presentations to create an environment most closely resembling the true practice of research discussion. Certainly, at the same time, the instructors should make some comments and give some suggestions to help students solve some problems or overcome some difficulties. This part of the course greatly encourages students to express their ideas and raise questions.

By having students choose different projects, they are more active and willing to communicate and discuss their work with other groups. For example, in this course, there were some groups that did not use the technique of SOE PCR or mutagenesis, but they still learned the principles and techniques by asking questions and via discussion. Class discussion proved to be highly useful and efficient for all students. This also expands their knowledge and improves their eloquence, which is important for an excellent scientist.

SOME PROJECTS INITIATED BY STUDENTS IN THIS COURSE

There have been several projects initiated by students each term. Some examples, listed in Table I, show that the projects were somewhat diverse, but mainly focused on the research of molecular biology. Most of the projects finished bioinformatics analysis, RNA isolation, RT-PCR, gene cloning and identification, and protein expression. Besides, some groups got mutagenesis or constructed a series of plasmids with different deletions. It would be considered to be successful if most of the above were finished in such short period. In terms of results, only the project Bioinformatics analysis, cloning, and protein expression of IL-18 gene did not get a specific PCR product and gene clone. This was because inappropriate tissue was chosen, and so the group failed to obtain the target gene, but they did a relatively good job in other aspects, such as bioinformatics analysis, RNA isolation, oral presentation, problem analysis, and final report.

Table I. Some projects designed and carried out by students
ProjectsSome resultsRemarks
Bioinformatics analysis, cloning, and tissue distribution of a novel renalase homologue from mouse.Gene basic information, RNA isolation, gene clone, tissue distribution, protein expression, and cell localization.Continued in research laboratory.
Bioinformatics analysis, cloning, and protein expression of IL-18 gene.Gene basic information, RNA isolation. (Failed to get gene clone.) 
Study on the expression characteristics of normal and mutation forms of human Apolipoprotein A-I gene (apoA-I) in E.coli.Gene basic information, gene clones of both normal and mutation forms, protein expression.Continued in research laboratory.
Bioinformatics analysis, cloning and protein expression of leptin gene (ob)Gene basic information, RNA isolation, gene clone, protein expression. 
Cloning, protein expression, and purification of a group of nonstructural proteins (Nsp) of mouse hepatitis protein.Gene basic information, RNA isolation, 14 gene clones, three proteins to be expressed and purified.Continued in research laboratory.
Bioinformatics analysis and cloning of a novel undifferentiated embryonic cell transcription factor 1 (UTF-1) from F9 cells.Gene basic information, RNA isolation, gene clone.Continued in research laboratory.
Bioinformatics analysis, molecular cloning, and protein expression of heptocellular carcinoma related protein 1 (HCRP1).Gene basic information, RNA isolation, gene clone, protein expression.Continued in research laboratory.
Cloning, protein expression, and purification of the spindling-like protein 2 (SPIN2) gene with N-terminal deletion.Gene basic information, gene clone with N-terminal deletion, protein expression, and purification.Continued in research laboratory.
Cloning and study on the structure and function of a stem cell pluripotency factor, Nanog N-terminal.Gene basic information and two gene clones with different deletions for testing their transcriptional activities.Continued in research laboratory.
Phosphatylserine receptor as a potential transcription factor: its N-terminal and C-terminal conserved domains are involved in its regulatory role on Rex-1 promoter.Gene basic information and two gene clones with different deletions for testing their transcriptional activities.Continued in research laboratory.
Developmental expression patterns and molecular cloning of mouse spermatogenesis associated 4 (SPATA4) gene.Gene basic information, RNA isolation, gene clone, and developmental expression patterns at three stages of mouse.Continued in research laboratory.
Bioinformatics analysis, cloning, protein expression, and purification of melanocyte proliferating gene 1 (myg1) gene.Gene basic information, RNA isolation, and gene clone. 

After the course ended, most of the projects were continued in the research laboratories. Although these projects only produced some preliminary data, they laid a good foundation for further study on the functions of these genes and provided very good training for the students. To avoid some publication conflicts, here we only show some data (Figs. 1–5) from one project, Bioinformatics analysis, cloning, and protein expression of leptin gene (ob), because the leptin gene has been well studied and this project has not been continued in the research laboratory.

Figure 1.

Bioinformatics analysis of the gene. (a) Chromosome localization. The mouse leptin gene is located on chromosome 6A and its mRNA covers three exons. (b) Protein structure. The coding sequence encodes 167 amino acids and has a typical leptin conserved domain. (c) Alignment of human and mouse leptins. The data indicate that the human and mouse leptins share high similarities.

Figure 2.

Amplification of the target gene. (a) RNA isolation. Adult mouse fat was used to isolate total RNA by TRIzol reagent (Invitrogen). 28S and 18S bands showed that the RNA was of good quality. (b) Amplification of the leptin gene. The amplified specific fragment by RT-PCR (TaKaRa One Step RNA PCR Kit) was about 0.5 kb, which is in accordance with the leptin gene. M: DNA markers.

Figure 3.

Diagram of the constructed expression plasmid. The leptin gene fragment and pGEX-4T-1 vector (GE Healthcare) were digested by BamH1+Xho1 (TaKaRa Biotechnology Co.) and ligated by DNA T4 ligase to form the plasmid pGEX/leptin that expresses GST/leptin fusion protein.

Figure 4.

Identification of the clones. To identify the positive clones, the miniprepped plasmids were digested by BamH1 and Xho1 enzymes. All picked-up clones were verified to have the target genes. Lanes 1–3: positive clones; M: DNA markers (New England Bio-Lab).

Figure 5.

Protein expression. The pGEX/leptin plasmid-transformed BL21 bacteria were induced by 1 mM IPTG (Sigma). The cells were lysated and resolved onto 10% SDS-PAGE. M: protein markers; Lane 1: whole cell lysates before induction; Lanes 2–5: whole cell lysates at different hours after induction. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

POSSIBLE PROBLEMS AND SOLUTIONS

To avoid frequent trouble in molecular biology techniques as much as possible, and ensure its feasibility for being a course, our instructor team reviewed all proposals carefully and gave students some suggestions in advance. In addition, when students encountered some trouble while performing the experiment, instructors would give them some suggestions in time. For example, when one group had some trouble in amplifying their target gene by trying different PCR conditions, we encouraged them to combine some other practical techniques, such as combining SOE PCR, because they were trying to amplify a relatively large gene. They got it by this improvement. By doing this, the main trouble spots were significantly reduced. Overall, this course raises the students' enthusiasm for laboratory work and let them get good training. However, the following problems should be emphasized:

  • 1)Some students may choose genes that are expressed only in certain conditions, such as diseased or stressed conditions. In this situation, it is a little bit difficult to obtain the gene's clone. To ensure students finish this course, the instructors can have several spare projects available.
  • 2)Some students design wrong primers because they have little experience in this aspect. This means that the PCR or cloning may fail, which can frustrate or discourage the students. To avoid this kind of problem, the instructors must review all students' proposals carefully, especially primer design.
  • 3)Setting the PCR conditions is a problem that students may encounter. They sometimes fail to get PCR products by traditionally calculating the primers' Tm. In this case, the students are allowed to optimize the PCR conditions by exploring the gradient annealing temperature.
  • 4)This course costs a little more than a routine laboratory course, because the students are allowed to fail and repeat some experiments. Therefore, the budget must be planned in advance according to the students' proposals considering all these factors.

CONCLUSION AND DISCUSSION

Molecular biology techniques have become an essential part of the whole life science field. After taking this course, students not only gain more skills, but also learn how to plan and effectively implement a research project related to molecular biology. This course avoids exposing students to a set of techniques only for the sake of learning the techniques themselves. Writing a proposal provides them freedom and requires them to use their judgment in choosing their project. Bioinformatics analysis provides them a powerful tool for predicting the structure, function, conserved region, and similarity of genes or proteins. Primer designing provides them the concepts of the ORF and fusion protein expression. Their exposure to RNA isolation, RT-PCR, and DNA recombination provides them the skills of cloning a gene from a natural organism. Experience with protein expression, purification, and SDS-PAGE provides them a set of basic skills for protein manipulation. Working with mutation, deletion and tissue distribution provides them some primary ideas to study the gene function. Thus it can be seen that this course systematically combines computer work with a variety of biological experiments to create an environment that closely resembles the true practice of research.

Educators have explored many excellent ways to improve education and instruction, including problem-based learning or the inquiry-based problem-solving format, which has been proven to be effective [6–10]. The teaching philosophy within the undergraduate laboratory courses also has been shifted away from cookbook experiments toward guided inquiry [11]. I agree that an excellent teacher should help the students learn in ways that are sustainable and substantial, and that have a positive influence on how students think, act, and feel [12]. This course is designed for advanced learning, and provides the students a training platform before they begin their real research work. All of these skills help the students understand the fundamental concepts and build their confidence, and are especially beneficial in preparing scientists for professional careers [13]. During this training course, the students study many techniques, and learn how to synthetically use them to conduct research and reach their goals. These techniques cover bioinformatics analysis, RNA isolation, RT-PCR, TA-cloning, subcloning, plasmid manipulation, enzyme digestion, competent cell preparation, gel electrophoresis, protein expression, point mutation, deletion, and overlapping PCR, as well as many others. All these techniques are in common use in real research laboratories. Moreover, the oral presentation and the class discussion components of the course get the students more involved in the class. More importantly, the students are allowed to practice writing proposals and solving problems on their own. This not only gives the students an idea of how research is carried out, but also helps improve their acumen, preciseness, persistence, and creativity in scientific research. Students who have taken this course were all very satisfied with it. Most students reported feeling confident in their knowledge and skills of molecular biology, and summarized substantial gains in critical thinking, motivation, patience, independence, creativeness, communication, and friendship. The students worked much harder, and they were more focused on and enthusiastic about their self-designed projects than projects arranged by the instructor. Some students showed truly exceptional quality in scientific research. Their ability to solve problems was significantly improved. For example, when doing TA cloning, some groups successfully got positive clones on the first try, while other groups failed. In this situation, the students became very active in solving the problem. Through discussion and analysis with other groups, they found that the reason was simply that they had not thoroughly mixed the ligation buffer. After finding this solution, all the other groups successfully got TA clones (except the group mentioned earlier that failed to get the PCR product). Although this was a small case, it indeed played a very important role for the whole project. More importantly, during the experiments, the students were more active to discuss their problems and sought some references, which is seldom to occur in basic course or other courses made of some inconsecutive individual experiments.

This laboratory course was popular with the students, but there is always room for improvement. The following suggestions should be made to ensure more effective implementation: 1) This course should be offered to students who have just finished the basic Molecular Biology Laboratory course and have taken the course of molecular biology theory; 2) The course should take advantage of the designed integrated experiments from the basic course in order to save time; 3) The students should be encouraged to continue their projects when they enter the research laboratories. By doing these, not only will the techniques be integrated, but also the students' time and ideas will be integrated so that they can have more time to extend their projects, which will greatly help undergraduates realize their ideas within a limited time. As a matter of fact, after getting more support in the research laboratories, some projects initiated from this course have been completed and published [14].

Acknowledgements

I thank my laboratory assistant (Yingzi Li) for ordering all the reagents, my teaching assistants (Gu Jing and Xunhao Xiong) for their technical help, and all of my students for their hard work, especially Rongrong Hu, Wanxing Chai, Lixia Jin, Dian Chen, and Shaoling Qi for their data.

  • 1

    The abbreviations used are: PCR, polymerase chain reaction; ORF, open reading frame; SOE, splice overlap extension.

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