Web-based learning support for experimental design in molecular biology: a top-down approach.

An important learning goal of a molecular biology curriculum is the attainment of a certain competence level in experimental design. Currently, undergraduate students are confronted with experimental approaches in textbooks, lectures and laboratory courses. However, most students do not reach a satisfactory level of competence in the designing of experimental set-ups. This article describes the development of web-based cases that offer students the opportunity to practice their design skills while addressing realistic research questions. Even though this may seem obvious, the authors did not develop a virtual lab that is as realistic as possible. In such a lab, students automatically focus on issues that are beyond the scope of an undergraduate course, which may hamper the acquisition of the essential basic concepts. Instead, an approach was chosen in which students have to design the overall procedure first and work out the individual steps afterward. Especially during the first stage students' control was limited to shield them from practical complexities. The material was evaluated in a regular educational setting and it fulfills most of the requirements initially set. In the discussion, the developed format is compared with those that are used in other disciplines.

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In line with the Bologna declaration (1999), Wageningen University converted its education to the BSc-MSc system. Therefore, educational programs had to be revised. This opportunity was taken to reflect on a number of learning goals in the molecular biology curriculum. One of these goals is the attainment of a certain level of competence in designing an experimental approach. This implies the application of various techniques to address a specific research question. After finishing their undergraduate courses, the students usually know how different techniques work. However, they have difficulties in selecting a set of appropriate techniques to solve a specific problem. Furthermore, working out techniques in more detail and tailoring them to specific situations, as well as combining different techniques, appears to be a problem. Last, students do not accurately use their knowledge of biology when designing an experimental approach. Thus, students do succeed in acquiring knowledge of different techniques as well as of biology during their undergraduate courses, but they have difficulties in applying this knowledge actively for the design of experimental approaches.

The indicated problems in experimental design may be inherent in the traditional educational setting. There, students are confronted with experimental approaches in textbooks, lectures, and laboratory courses. Each of these formats has it's own drawbacks. Textbooks do describe many experiments and approaches, but this is not sufficient for students to learn how to choose techniques and how to schedule operations. One of the problems is that students usually focus on the technical details of techniques, thereby losing sight of the uses of such techniques. In a lecture, more weight can be put on the actual designing of experimental approaches. It is however, very hard to involve more than a limited number of students in such a way. Furthermore, the students who do get involved can hardly get personal feedback due to considerations the lecturer has to take of the other students as well as of the time allotted for the lecture. Finally, in a laboratory course, students have little freedom in choosing and scheduling operations due to a range of practical limitations. In addition, carrying out poorly designed experiments may cause too much frustration. Moreover, during a laboratory course, students become preoccupied with the practical skills they still lack, such that the overview of even the specific experimental approach they follow themselves is lost. Thus the teaching of more general aspects of designing experiments is hardly possible. The possibilities of computer based learning support may offer a solution to the problem. Apart from the well known argument that computer based learning support makes it possible to activate each student individually and generate personal feedback, many of the results of molecular biology experiments can be represented digitally. This offers, in principle, the opportunity to let students perform experiments virtually and show them the resulting experimental data. These experiments in particular include those that cannot be performed in real laboratory courses due to practical limitations as well as badly designed ones. Trying out badly designed experiments virtually may be very instructive and much less frustrating than performing them in an actual lab. Posner and Rudnitsky (1997) also stated that significant learning often occurs in a setting where it is safe to try.

In molecular biology research, a standard way to obtain information about the function of a gene, is by way of the creation of genetically modified organisms in which this gene is overexpressed or disrupted. With the recent sequencing of several complete genomes, it is expected that the usage of this approach will increase even more to translate this DNA sequence information into functional information (Knight & Abbott, 2002). Therefore the authors decided to create digital cases in which students have to design a procedure to create such genetically modified organisms. They also carry out the experiments virtually and interpret their results to design the subsequent steps of the approach.

This article describes the development of these digital cases, including the requirements that have to be met, a description of the actual material as well as evaluation results. Eventually, a format was developed for the cases, in which students have to design a procedure in a top-down fashion. In the discussion, this format is discussed further and it is compared with formats that are used to teach design in other fields.

REQUIREMENTS FOR THE DIGITAL CASES

In the regular course, the cases are used to supplement lectures and a textbook (Lodish, Berk, Zipursky, Matsudaira, Baltimore, & Darnel, 2000). The material is useful in this setting, when a number of requirements are met.

First, the students should appreciate working with the material. The opinion of the students is assessed through evaluation forms. They should judge the material with at least a 7.5 (scale 1-10) on average. Furthermore, the material should be really instructive in their perception, they should enjoy working with the material, and they should prefer working with the material above getting additional explanations during lectures. In disagree-agree questions of this nature, they should give a score of at least 4 (scale 1-5) on average. Wageningen University assesses the students' perception of the quality of courses, course material and teachers on a regular basis with standard evaluation forms that consist of disagree-agree questions. An average appreciation of 3 on a scale of 1 to 5 on these forms is considered satisfactory and an average of 4 or more results in a letter of praise from the university. Therefore, the authors are satisfied when students give a score of 4 or more on a scale of 1 to 5.

Second, students should not only appreciate working with the material, they should also develop their experimental design skills by working with it. To get an indication of this, exam results are analyzed. In the exam a number of questions are included that deal with selecting and working out techniques for the creation of genetically modified organisms. The students should get a score of at least 7 (scale 1-10) on average for these questions.

Apart from these requirements for the regular setting, the material should also meet another requirement. As the quality of learning material is partially determined by the extent to which the material is exposed to critique from peer reviewers and students, it is important that the material not only be used in the regular educational setting, but also in several different educational settings in different places. To ensure this, the cases should form modules that are fairly independent from each other, so a student or teacher can select the cases he is interested in. Furthermore, the material should in principle be suitable for self-study. Moreover, the material should be able to support students with different degrees of prior knowledge. Finally, to make sure that the material can also be used at other universities, it is important that it is world wide accessible.

FORMAT OF THE CASES: STUDENTS DESIGN AN EXPERIMENTAL APPROACH IN A TOP-DOWN FASHION

To simulate an actual research situation, the authors initially wanted to create a virtual laboratory in which students have the opportunity to perform a range of different experimental steps. A step can be carried out virtually after selecting it and designing it in more detail. By interpreting the experimental data, the student can find out whether or not he made good decisions. In such a virtual lab, the student has to actively use his knowledge of techniques and of biology in a situation in which he has a lot of control and ample opportunity to test his own ideas. However, after analyzing the student's task in such an environment in more detail, it was concluded that this format is not really suitable for undergraduate students. In the described virtual lab, the student basically has to design at two different levels simultaneously: he has to design an overall procedure by assembling several experimental steps and he has to work out these individual steps in more detail, while making use of results from previous steps. Students should be capable of designing at this latter detail level while keeping track of the overall strategy. Moreover, it is very useful to practice this, because it requires active integration of knowledge of biology with knowledge about techniques. For the designing of the overall procedure itself, the situation is different. In molecular biology, procedures are not simply right or wrong. Instead, they can be placed on a scale of more or less useful. To judge the precise usefulness of an experimental procedure, knowledge of a range of practical issues such as efficiency, sensitivity, and reliability is required. This knowledge is usually only acquired after extensive practical experience and this is beyond the scope of an undergraduate course. However, in the initially proposed virtual laboratory, students may have to compare different alternatives that are so equivalent, that this practical knowledge is required to select one of them. This may distract students from the more important conceptual issues and it can lead to premature cognitive overload. Thus, such a virtual laboratory does not fully support the intended learning goals for undergraduate students, because students' control is too high for the designing of the overall procedure. Schwier and Misanchuk (1993) also caution that only those learners who are generally high achievers or who are knowledgeable about an area of study can benefit from a high degree of learner control. To really focus on the learning goals for undergraduate students, another format had to be developed in which the student gets less control for the designing of the overall procedure. At the same time however, the student should keep an overview of what he is doing, so he should not just simply follow instructions and carry out experiments without knowing why.

The fact that in this case it is actually only possible to work out individual steps in more detail once the whole procedure is known, inspired the development of a format in which the student is first supported to design the overall procedure and only once this procedure is designed, can he carry out the experiments virtually. Thus, students design the procedure in a top-down manner. Students' control can be different in both phases. By limiting students' control in the overall design phase, they can be shielded from unnecessary complexities. Moreover, as the overall procedure is designed before the student actually performs it, it is also possible to include additional support while the student is performing the experiments. Thereby, questions can be included that address the interpretation of specific aspects of an experimental result as well as questions that highlight the possibilities of alternative techniques. As the student designed the procedure himself however, he should be able to keep an overview instead of following the instructions blindly. In subsequent cases, students' control can gradually be increased at the two levels independently.

EXAMPLE: A CASE ABOUT THE CREATION OF A "SMART" MOUSE

One of the developed cases deals with the creation of a "smart" mouse and can be viewed at the demo site (http://mbedu.fbt.eitn.wau.nl/demo_jilr). Upon starting the case, the student gets a short introduction about memory formation and he receives the assignment that he has to create a specific transgenic mouse to find out whether a certain protein is involved in learning. The subject by itself is not relevant: it merely serves to motivate the student. Furthermore, based on the Case Based Reasoning (CBR) theory (Schank, Berman, & Macpherson, 1999) learning will be promoted by working out a realistic research question. After reading the introduction, the student can start designing the outline of the procedure. In this case, the student first has to answer several multiple-choice questions that support him in analyzing the assignment. Furthermore, the student has to choose between two general overall procedures after which the conceptual differences between these alternatives are highlighted. Then the student has to work out this general procedure further by selecting a number of experimental steps and putting them into order (Figure 1). Theoretically about 0.4 million different procedures could be proposed, so it is impossible to simply scan each possibility and select the most useful one. The steps the student can select from are chosen in such a way that the student should be able to design a procedure by actively using the background knowledge he has. Thus he is shielded from a number of practical complexities. In this case, the student does not get the option to work with second-generation offspring of the initially altered mouse (F2 mice) for example. A part of these F2 mice are homozygous for the additional gene and to work with them has some practical advantages. However, generating these mice requires additional time. By not offering the possibility to work with F2 mice, the student does not have to consider these practical issues and can focus on conceptually more important ones instead. Upon submission of the proposed procedure, the student receives feedback that depends on the procedure he proposes and that gives hints to further improve the proposed procedure. The feedback is generated by the program on the basis of a form of pattern matching. The student could for example propose to test the learning capabilities of a mouse after determining the expression levels of a receptor in the brain of the same mouse. He then receives as feedback that this order is not useful as it is necessary to obtain a sample of the brain for the determination of the expression and that, even if the mouse would survive this procedure, this will certainly influence its learning capabilities. This example also illustrates that the student should use his knowledge of biology: he should realize that gene expression levels differ from tissue to tissue. In this case, using the feedback eventually leads to the design of one single procedure. Because of the direct availability of feedback that is dependent on the student's answer, the procedure can be designed by students with varying levels of competence. Students with a relatively low level of competence for the skill will perform more iterations thus receiving more hints and support.

[FIGURE 1 OMITTED]

Once the outline of the experimental procedure is designed, the student can perform the different steps virtually. Figure 2 shows for example the experimental result of a southern blot analysis. To perform this analysis the student had to select the restriction enzymes he wanted to use. The program then calculated the position of the bands and generated the experimental result. The student has to interpret this result by answering several questions. These questions also help the student in determining whether he chose a useful restriction enzyme combination. In the figure, the student has to determine how many copies of the added gene construct were inserted into the genome of the mouse. Therefore, he has to realize that the added gene is also already present in normal wild type mice and that the presence of this wild type gene is responsible for (only) one band. This, in turn, is only possible when knowledge of the southern blotting technique, of the overall procedure and of biology are actively integrated. In this case, the student chose a useful restriction enzyme combination so that it is indeed possible to determine the copy number in mouse 9 (which is 2).

[FIGURE 2 OMITTED]

Instead of southern blot analysis, students often propose to use another technique, the polymerase chain reaction (PCR) technique, to determine the copy number. Therefore, upon completion of the southern blot analysis, the student is asked whether PCR could have been used instead. He then finds out that this is not the case. Thus, in this way the conceptual differences between southern blot analysis and PCR analysis are emphasized in a context in which these differences are relevant. After answering this question, the student can continue with the next experimental step. In case the student needs the results of the southern blot analysis when performing one of the later experiments, he can easily access an overview that shows his personal results of the analysis. By performing all virtual experiments, the student can discover that overproduction of the protein in mouse brains does indeed lead to improved learning and memory formation.

After finishing the virtual experiments, the student gets some additional information in which the case is briefly compared with the original research paper (Tang et al., 1999). This stresses the relevance of the technique for current research, which should further motivate the students, as being aware of the relevance of theory is an important motivational factor (Keller, 1987). Moreover, some additional philosophical comments are given about the reasons why organisms do not naturally have higher levels of the studied protein in their brains. This should stimulate the student to think further about the content. Afterward, the student can view a summary, which shows his personal results. This also contains all the theory the student has to master, including information about alternative options the student did not necessarily select himself. This also serves to prevent disadvantaging students who went through the cases without making any mistakes, which is a potential problem that is also discussed by James (1998). The case is completed by a self-test with questions about the most important concepts that were dealt with in the case.

Some background information the student may need to go through the case, can be found in a separate library that can be accessed at any time. Providing learner-selectable information just-in-time is, for example, recommended by Jonassen (1999).

EVALUATION

Evaluation of the material in the regular setting

The first two developed cases about genetically modified organisms were evaluated in our regular course. Nearly all students have enough biology background knowledge and they have some practical experience with a number of basic techniques. As we previously received indications that the awareness of being a participant in a study makes students more motivated, the students were not informed about this and the evaluation was carried out in a completely regular setting. Students only had to fill out an evaluation form in the end, which is not uncommon for them. A disadvantage of this approach is that the test situation is less controllable. In this case for example, a number of students who did work with the material, did not make the exam questions about it. In total 41 students filled out the evaluation forms and 35 students answered the exam questions about the creation of transgenic organisms.

The evaluation forms were used to assess the judgment of the students. The results are outlined in Table 1. It must be noted that this overall judgment also includes a case with another format, which has not been described in this article. As the authors did not receive any indications that the students judged this additional case more positively than the ones described in this article, it was concluded that the design requirements concerning the judgment of the students, were fulfilled.

To get an indication of the level of competence attained by the students, the exam results were analyzed. The students scored very well for the part of their exams which deals with the creation of transgenic organisms (Table 2) and the requirements were indeed met for most of these questions. The students scored at least 7, except for question 3. This question indeed requires the application of a relatively difficult concept: the students have to realize that they have to make use of unknown restriction sites outside the construct. In both digital cases, students were confronted with a problem that is similar to the one they have to solve for exam question 3. To get more insight into the learning process, tracking data were analyzed. These tracking data indicate which pages were opened on which computer. Even though it is not known which student(s) were working with a certain computer and whether they were simply guessing or not, these data may still give a rough indication of the process. When the concept had to be applied in the first case, on 15% of the computers, the first given answer was correct. In the second case, this happened on 29% of the computers. At the exam, 60% of all given answers were good enough (worth a score of 6 or more). This suggests that every time the concept has to be applied, some more students actually grasp it. Students who do not master the concept after going through the cases may have given the right answer by coincidence and may not have realized why their answer was correct. To improve the results in future, feedback will be included that explicitly states why the chosen approach is useful. Furthermore, in an additional case that was developed later the students have to apply the concept again, which may also lead to better results. Thus, the students scored very well for their exams and the requirements concerning the exam results are nearly satisfied. The authors expect the cases will fully meet the requirements for the regular setting once the proposed improvements are implemented.

Usefulness of the Material in Different Educational Settings

To promote the usefulness of the material for different educational settings, a number of additional requirements had to be satisfied. First, the cases had to form fairly independent modules. Therefore, there are no cross references between different cases. Furthermore, each case starts with an introduction and finishes with a self-test, thereby forming an independent learning unit. Moreover, even though subsequent cases are increasingly difficult because students get increasingly more control, it is not assumed that students went through the previous cases. If necessary, students can still receive basic feedback and they can look up all background information in the separate library. To facilitate the usage of this library, it is always easily accessible and when there is information present in the library that may be helpful to solve a certain problem within a case, this it is indicated and a direct link toward the specific information is provided. Furthermore, the library has a very modular structure as well, so that it is relatively easy to study a specific part of the theory only. The cases also had to be suitable for self-study. The implemented feedback and the background information in the library are also useful for this purpose. It is of course not always possible to predict whether the given information is sufficient or not. Therefore, students' questions during tests are documented and this information is used to make the site more self-explanatory. The included feedback and the library objects also make the material more suitable for students with varying degrees of prior knowledge. It often depends on the answer of the student how basic the information in the feedback is. Thus, students only receive the very basic information when they need it, so that those who do not show they need it, do not get bothered with it. The practical aspect of the requirement that the cases can be useful in different universities has lead to the decision to make the material completely web based. Thus, the usefulness of the material for different educational settings was promoted in several ways.

We have some preliminary indications that the material is suitable for different educational settings. First, the material was also used by six students who did not have the required biology background and who did not have any experience with molecular biology techniques. Despite their lack of prior knowledge, they gave the material an overall score of 7.8 (scale 1-10) on average and they indicated that the level of the material was good for them. Second, some students used the material at home and managed to go through the cases without additional help from teachers. Third, two students used a case about the creation of a salt tolerant tomato plant to learn something about making transgenic plants while they participated in an elaborate advanced practical course. They studied the case in the laboratory during some waiting steps of their experiments. It would not have been possible for them to study this case separately, if the cases would not form independent modules. Thus, there are some indications that the material can be used in various situations. The plan is to perform additional evaluations to yield more reliable data concerning the suitability of the material for different educational settings.

DISCUSSION

To improve the experimental design skills of students, a set of digital cases was developed. It was decided not to simulate a real lab as realistically as possible, because students are automatically confronted then with issues that are beyond the scope of an undergraduate course. Instead, a format was developed in which students design the overall procedure first and work out the individual steps afterward. Thereby, students' control can be limited in both phases independently. Especially during the first phase, students' control was limited in order to shield them from certain practical complexities. An obvious disadvantage of this format is that students do not learn to take these practical factors into account. On the other hand, this format offers the advantage that it is possible to really focus on specific learning goals, so students can learn the underlying concepts relatively fast. This, in turn, should form a good basis to further develop one's design skill later to a level at which these practical complexities are taken into account as well.

Besides molecular biology, there are also other disciplines in which design essentially implies designing at an overall level and designing at sub levels simultaneously. Bridge, a tutorial environment for novice programmers (Bonar & Cunningham, 1988), is an example of a program with a format that is similar to the one described in this article, even though the subject matter and the design issues involved are very different. There are also other formats that are employed to learn design. They offer students different degrees of control. For an introductory course in process engineering for example, a design environment was developed in which students have to design chains of unit operations and where they have to adjust parameters for each operation (Van der Schaaf, Vermue, Tramper & Hartog, in press). In this environment students' control is higher than in the environment that was described in the current article. To make sure that the inexperienced students for whom the material is intended, do indeed benefit from the environment, the students are given assignments starting at a relatively low level and becoming increasingly complicated. A format with another nature, the completion strategy, was developed for an introductory course in programming (van Merrienboer & Krammer, 1989). In the completion strategy, students have to complete well-designed, incomplete programs while making use of worked out examples. To be able to complete the program, the student has to understand the given overall structure. Compared to the format that was described in this article, the completion strategy obviously offers the student less control for designing the overall structure, but at least as much control for designing at sublevels. The preferred format is determined not only by the precise level of the students and the specific learning goals, but also by the nature of the subject. For example, the format that was developed for process engineering could not have been used because the simplest procedure to create a genetically altered organism is already fairly complicated. On the other hand, when the outcome of a single step, for example, largely determines the nature of the next one, designing in a top-down manner is probably not possible, nor desirable.

Designing experimental approaches is a complex cognitive skill students do not sufficiently master during their undergraduate years in the traditional educational setting. To tackle this problem, highly interactive digital learning material was developed, which provided personal feedback and generates experimental results depending on the student's decisions. With this material, students can practice to apply their theoretical background knowledge for experimental design without great time investment. Because of the good experiences thus far, the plan is to use web-based learning materials to support other complex cognitive learning goals that are hard to achieve in the traditional classroom.

Table 1. Results from Evaluation Forms (n=40)

The given marks are compared with the marks that were required in
advance. A statistical outliner ([greater than or equal to]6 SD from the
mean) was discarded. Including it would not alter the conclusions
concerning the requirements.

       Evaluation question         Mark  Required

                                     scale 1-10
Give a mark (scale 1-10) for
  the computer part of the course  7.8   [greater than or equal to] 7.5
                                     1 (disagree) -
                                       5 (agree)
I liked working at the site.       4.1   [greater than or equal to] 4.0
I learned a lot from working
  at the site.                     4.1   [greater than or equal to] 4.0
I prefer working at the site
  to additional lectures.          4.3   [greater than or equal to] 4.0

Table 2. Average Scores for a Number of Exam Questions (n=35)

Obtained scores are given on a scale of 1 to 10. A score of at least 7
was initially required for each question.

Introduction

It is hypothesized that the T-bet protein is involved in regulating the
balance between two types of T-helper cells and that a surplus of T-bet
causes autoimmune diseases. To test this hypothesis, you decide to
create a transgenic mouse in which the T-bet gene is overexpressed in
the whole body. Therefore, you use the following construct (the
CMV-promoter originates from the Cytomegalo virus).

                      Question                                     Score

1 Describe the procedure you would use to create the transgenic
  mouse.                                                            8.6
2 How can southern blot analysis be used to distinguish the
  transgene from the endogenous T-bet gene? Indicate the position
  of the probe(s) you would use.                                    8.3
3 Which restriction enzyme(s) would you use to determine the
  number of integrated copies when southern blot analysis is
  used? Make also a drawing of the autoradiogram for control mice
  and transgenic mice that contain two copies of the transgene.     6.2
4 Is it possible to use PCR to distinguish transgenic and
  non-transgenic mice? If so, indicate the positions and
  orientations of the primers.                                      7.2
5 How do you determine whether T-bet is overexpressed in white
  blood cells?                                                      7.7

Acknowledgements

We would like to thank Olivier Sessink and Bert-Jan de Hoop for technical implementation, Gerard Moerland for multimedia support and Martin Mulder and Harm Biemans for providing pedagogical information.

References

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