In an effort to achieve high success in knowledge and technique acquisition as a whole, a biochemistry and molecular biology experiment was established for high-grade biotechnology specialty students after they had studied essential theory and received proper technique training. The experiment was based on cloning and expression of alkaline phosphomonoesterase (AP) from Escherichia coli JM83. The strategies undertaken to deal with the failure of individual experiments and to ensure each experimental group get along well are discussed here. A reasonable evaluation system has been established to improve the effectiveness of teaching, which includes four items: self-evaluation, a group presentation, work on duty, and experimental reports. These items were monitored for contribution to the students' perceived cognitive achievement. Motivation for learning seems to have been improved in the laboratory, particularly by the involvement in a presentation of the experimental work and results.
Biotechnology specialty students in East China University of Science and Technology (ECUST) have taken basic theory courses, such as essential biochemistry, microbiology, and cell biology, and received basic technique training, such as basic biochemical and microbiological technique training in the first 2 years of 4-year undergraduate system (Table I). There is also 1 year's scientific training in 4th year waiting for them to fulfill their graduation thesis (Table I). However, in 3rd grade, they are still unable to fully understand the combination of knowledge and technique acquisition as a whole. Therefore, we developed a comprehensive biochemistry and molecular biology experiment 5 years ago. Various problems were encountered during the progress of the experiments. For example, the biggest problem was the failure of individual experiments for some student groups, leaving them without proper samples for subsequent experiments. Strategies to solve these problems have to be provided. Furthermore, an evaluation system has been established to improve the effect of biochemistry and molecular biology experiment teaching. After several years' practice, it has been proven that the biochemistry and molecular biology experiment could provide high success in knowledge and technique acquisition as a whole. The students' scientific research capabilities and interest in the biotechnology specialty have been improved.
Table I. The main courses and experiments for biotechnology specialty students in ECUST
Basic chemistry, basic physics, advanced math, physiology, etc.
Basic chemical experiment, basic physical experiment, and physiological experiment
Organic chemistry, basic biochemistry, cell biology, basic genetics, microbiology, etc.
organic chemical experiment, basic biochemical experiment, basic genetic experiment, and microbiological experiment
Molecular biology, proteomics, genomics, glycobiology, etc.
cell biological experiment, biochemistry, and molecular biology experiment
General technical training in factories (1 mo), specialty scientific training to fulfill bachelor thesis in a research laboratory (about 1 yr, each student selects one's favorite research group)
EXPERIMENT CONTENTS AND PROCEDURE
The biochemistry and molecular biology experiment focuses on the cloning and expression of the alkaline phosphomonoesterase (AP) gene from Escherichia coli JM83, including the isolation of the specific DNA fragment encoding AP from the genomic DNA of E. coli, with the usage of the PCR technique, subcloning of the DNA fragment into a plasmid so as to make a construct that will overexpress the protein in E. coli cells, and SDS-PAGE analysis of the expressed protein [1, 2].The time schedule and contents of the experiment are shown in Table II, and the procedure of the experiment is shown in Fig. 1.
Table II. The time schedule and the contents of biochemistry and molecular biology experiment
Chromosomal DNA extraction and PCR
Chromosomal DNA extraction; DNA electrophoresis; PCR of alkaline phosphomonoesterase (AP) gene
PCR product recovery, ligation, and transformation
PCR product electrophoresis; PCR product recovery; AP gene ligate to vector pMD-18T; transform ligation mixture to competent cells DH5α; culture overnight
Plasmid extraction and identification of pMD-18T-AP
Inoculate two or three clones and culture overnight; plasmid extraction; DNA electrophoresis; digest with EcoRI/SphI; DNA electrophoresis
Construction of expression plasmid pET-11a-AP
Digest both pMD-18T-AP and pET-11a with NdeI/BamHI; gel-recovering; ligate AP gene with pET-11a; transform ligation mixture to competent cells DH5α; culture overnight
Plasmid extraction and pET-11a-AP identification
Inoculate two or three colons and culture overnight; plasmid extraction; DNA electrophoresis; digest with EcoRI/SphI; DNA electrophoresis
Preparation of BL21 (DE3) competent cells and transformation
Preparation of competent BL21(DE3) cells; pET-11a-AP plasmid transform to BL21(DE3) competent cells; culture overnight
AP Expression and SDS-PAGE
Inoculate two or three clones and culture overnight; induction; SDS-PAGE
The seven experiments are detailed below, with notes on how to act if the experiment fails.
1) Chromosomal DNA extraction and PCR
Chromosomal DNA is extracted and confirmed by DNA electrophoresis, and then PCR is performed to obtain the AP gene using the chromosomal DNA as a template. If a student could not extract the chromosomal DNA or the result of the PCR is negative, redo.
2) PCR product recovery, ligation, and transformation
PCR product is ligated into vector pMD-18T for 2 hours at 16°C, and the resulting clone is then transformed into competent cells, which are cultured overnight.
If a student is not able to recover PCR product or if the PCR product extraction yield is too low to do a ligation, redo the experiment.
3) Plasmid extraction and identification of pMD-18T-AP
Two or three clones are then selected and cultured overnight, and the plasmid is extracted (each clone for each student) and digested with EcoRI/SphI.
If no clones grow on a student's own plate, he/she can select clones from another student's plate. Note: the sample is borrowed.
If the plasmid could not be extracted from one clone successfully, redo the extraction by trying another clone. Note: redo.
4) Construction of expression plasmid pET-11a-AP
Both plasmids, pMD-18T-AP and pET-11a, are digested with NdeI/BamHI after gel recovering of the corresponding bands and are then ligated for 2 hours at 16°C. The ligation is then transformed into competent DH5α cells, which are cultured overnight.
If only a very light band is observed and there is not enough DNA to perform a ligation after gel recovery, the student can borrow another student's DNA fragment or he can try his own sample.
If one could not recover enough DNA even though a suitable amount of plasmid is digested and there is a dark DNA fragment band after enzyme digestion, the teacher will help him to analyze the cause and encourage him to repeat the recovery procedure by either reloading or recleaning the column and will try to determine which step does not work.
5) Plasmid extraction and pET-11a-AP identification
Two or three clones are selected and cultured overnight. The plasmid is then extracted and identified with double-enzyme digestion with EcoRI/SphI.
If no clones grow on a student's own plate, select clones from another student's plate. Note: the sample is borrowed.
If a student could not extract the plasmid from a clone successfully, he could redo the experiment by trying another clone. Note: redo.
6) Transformation of BL21(DE3) competent cells
Competent BL21(DE3) cells are prepared and transformed with the pET-11a-AP plasmid.
7) AP Expression and SDS-PAGE confirmation
Two or three clones are selected and placed into culture tubes (containing LB medium and ampicillin) , and grown overnight. Then, 1% cultured cells is inoculated into another culture tube (containing LB medium and ampicillin), cultured for 3 hours. IPTG is added to a final concentration of 1 mM to induce expression, and the bacteria are cultured for another 3 hours. Finally, the samples are analyzed by SDS-PAGE.
If no clones grow on a student's own plate, select clones from another student's plate. Note: the sample is borrowed.
The biochemistry and molecular biology experiment includes cloning of a gene and expression of the corresponding protein and involves all the essential experimental parts, from genomic DNA template preparation to AP expression. Although this might be considered as a routine molecular biology experiment , it is unrealistic to ensure that every step works for each student. Thus, the biggest problem is failure of individual experiments. The failure may occur in all steps, here are listed some failure steps occurring often, for example:
1Not enough amount of chromosomal DNA is extracted.
2The result of the PCR is negative.
3No sufficient DNA for ligation after DNA gel recovery.
4No positive clones grow after about 14 hours' culture.
During the experiment, repeating an individual experiment is not always feasible. We have to set up reasonable strategies to deal with negative results and move on to the next experiment. Furthermore, an evaluation system is necessary to track and score the students on the whole procedure, not only on the final results of the experiment. The students are asked not only to perform the experiment properly but also to understand the principle of each step and to be able to analyze the results, whether they are positive or negative. For negative results, the students should know how to analyze and improve the results. Thus, students' analytical ability is another evaluation standard.
If the time allowed, the students failed in the first time experiment are encouraged to redo experiment, otherwise they may instead borrow a sample from a successful group to move on to the next part of the experiment. The students are encouraged to get the results of each step by themselves. Some points will be deducted in their final scores whenever they borrow samples from other groups.
After the teaching practice and gradual improvement for several years, an effective evaluation system has finally been established. The procedure of the assessments on the students' experiment includes three steps, as shown in Fig. 2. Step 1 is to perform an evaluation of the results of each unit experiment in which the students are asked to evaluate their results properly. Step 2 involves a team project in which the students prepare and give a presentation in class at the end of the experiment. Step 3 is two assessments from teacher to the students' work on duty and the experimental report. So, the composition of the evaluation system includes four parts: 1) Self-evaluation of the student's work toward the experimental results (negative or positive); 2) presentation about the experimental procedure, principles, results, and analysis; 3) work on duty; 4) the experimental report.
The students are asked to make formal scientific figures, with pictures of the results, based on their experiment results, including any failed results and paste them in Table III (page 273). The students are then instructed to evaluate their results properly. The teacher will evaluate their work and adjust the self-evaluation to give them a reasonable score.
Table III. Summary of self-evaluation on your own experimental results
Results (insert figures of your results here. If redid, insert two figures)
If borrowed, please note from which group you borrowed
Result analysis (if repeated, please note here and analyze the reason for failure)
Self-evaluate your score (A/B/C/D). If your results borrowed, please decrease one grade
Chromosomal DNA extraction
PCR product recovering
pMD-T-AP extraction and identification
pMD-T-AP and pET-11a digested with Nde I + BamH I
Recovering AP gene and pET-11a
pET-11a-AP extraction and identification
At the end of the experiment, there are experimental presentations. All the students in a class are divided into six or seven groups, and each group consists of four students. Each student should participate in preparing the presentation. One is in charge of giving the presentation, one acts as a judge, and two are in charge of answering questions posed by other group members. The students should clearly present the procedure, principles, results, and discussion of the experiment. If a step is failed, analysis of the negative results has to be presented, and if the experiment has been repeated, this should be explained in detail. The group of judges consists of students from each group, and each should promise to be impartial (Table IV).
Table IV. The standard scores given to a presentation group (only used by judges)
PPT (3 points)
Presentation (3 points)
Question reply (3 points)
Time limited (1 point)
Total (10 points)
PPT, three points will be given for the clear station of the procedure and principles (1′), proper format of results (DNA marker and the desired band were shown clearly) (1′), and reasonable analysis (1′); presentation, three points will be given for loud and clear speaking of the presentation (1′), clear convey of the principles, procedure, and results of the experiment (1′), and analysis of the whole experiment (1′); question reply, three points will be attained if the students, excluding the presentation, answer properly when at least three questions are raised; time, one point is given if the presentation is finished within 15–18 min.
Two experimental reports are submitted for each student at different levels. The report of the first level consists of the unit experimental reports, such as genomic DNA preparation, PCR, ligation, screening, and so forth. The report of the second level is a comprehensive experimental report, which is same as scientific experimental reports, integrating all parts of the experiments, including the aim, principles, procedure, results, and discussion.
Work on Duty
There are four students on duty in each group. An important responsibility of the students on duty is to help the teacher to prepare the experiment, including the preparation of the media, separation of samples into parts, preparation of the DNA electrophoresis gels for each group, handing out, and collection of samples. Another duty is to keep the laboratory clean.
Final Score Calculation
The final score for each student is calculated according to the above evaluation and the contribution of each part to the evaluation system, as showed in Fig. 3.
Course evaluations, completed by the students, represent an important informative measure of student satisfaction with the learning experience . The students evaluate the teacher and give some comments to the course. The average score for teachers participating in the experiment has been excellent in recent years. Quoted below are some comments from the students on the experiment:
1The experiment was interesting, and the teacher was able to explain the profound procedures and principles in a simple way, and let us know the key aim of each experiment.
2The biochemistry and molecular biology experiment allowed me to learn how to analyze problems and helped me turn knowledge into related scientific research.
3Repetition of the experiment gave me an opportunity to understand the problems and to learn more from failures.
4Giving a presentation in class was a challenge for me. I am happy that all members of my team were satisfied with my presentation.
5The evaluation system of the experiment is reasonable and just.
6My ability to perform scientific experiments has been improved.
There are also some negative comments on the experiment.
1Experiment lasted too long, sometimes I felt exhausted.
2I am so vulnerable that I am afraid to face to a failure result after one-day's efforts.
3I did not want to redo the experiment.
Response of Students to the Evaluation System
A questionnaire is designed to obtain students' responses to the evaluation system. In the questionnaire, students estimate the contribution of each of the four aspects of the evaluation system to their scientific training. These include self-evaluation, presentation, experiment reports, and work on duty. The results are shown in Fig. 4. The experimental presentation ranks first and self-evaluation is following. The experimental report, which is considered a main and important evaluating strategy in other teaching experiments, is not highly valued by the students here. Therefore, the students' perception is that the presentation contributes more to their understanding than the experimental report. Learning motivation may have been higher in the laboratory as a result of the involvement of each student in every step of the experiment .
The presentation takes place at the end of the experiment and forces the students to review the principles and the results carefully and critically once again. They devote themselves to preparing the presentation and reading references to analyze the results. Sometimes, the students can get additional materials from a search in the internet to show the principle of the experiment or to explain the experimental phenomena. Furthermore, they also enjoy sharing their experience of failure and success, and sharing different opinions from each group in the presentation and thus they can learn more from each presentation.
The biochemistry and molecular biology experiment provides greater success in knowledge acquisition for biotechnology specialty students. In 4th grade, it takes 1 year to fulfill the scientific training in laboratories for technological students. The students are asked to apply for entrance to their favorite research groups. Generally, they are welcomed by many professors. For their study of graduate thesis, 20–50% students find a place in some laboratories of Chinese Science Academy and some high-tech enterprises, and 39–50% students practice in State Key Laboratory of Bioreactor Engineering (ECUST). Most feedback from their supervisors is positive with good abilities in integration of knowledge and design, and operation of experiment. For example:
1They showed better abilities in knowledge acquisition.
2They can learn from the failure results and make a quicker response to analyze and correct them.
3They can analyze problems using the knowledge learned before or references to move the research on.
Another feedback is from the postgraduate student entrance interview. This is carried out at the beginning of the first semester of the fourth year just after they have undergone the biochemistry and molecular biology experiment training. When compared with the students from other specialties, the professors think that the students from biotechnology specialty show more self-confidence on some technical skills. In recent years, about 10% students are admitted as doctorial or master degree students in Chinese Science Academy or Fudan University, and about 15% students enter State Key Laboratory of Bioreactor Engineering as doctoral candidates. Two to five percent students are going abroad to do doctoral study.
Through the teaching practice of the past few years, an evaluation system has been established to give the students a reasonable final grade, which was also shown to be an effective way to improve the experimental teaching. The student presentation has been shown to be one of the most effective strategies of contributing to the students' understanding of the experimental procedures and principles. The adopted strategies solved the difficulties encountered during the process of the teaching experiment. These strategies include repetition of experiments if a particular experiment failed or borrowing a sample from other groups to ensure that the experiment is able to proceed. Positive feedback from the students and other teachers provides evidence of the significance of the experiment in increasing the students' interests and their scientific research abilities.