Q 2011 by The International Union of Biochemistry and Molecular Biology
BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 6, pp. 448–456, 2011
Laboratory Exercise A Linked Series of Laboratory Exercises in Molecular Biology s Utilizing Bioinformatics and GFP& Received for publication, May 24, 2011, and in revised form, August 6, 2011 Carey L. Medin‡ and Katie L. Nolin From the Department of Biology, Stonehill College, North Easton, MA 02357
Molecular biologists commonly use bioinformatics to map and analyze DNA and protein sequences and to align different DNA and protein sequences for comparison. Additionally, biologists can create and view 3D models of protein structures to further understand intramolecular interactions. The primary goal of this 10-week laboratory was to introduce the importance of bioinformatics in molecular biology. Students employed multiprimer, site-directed mutagenesis to create variant colors from a plasmid expressing green fluorescent protein (GFP). Isolated mutant plasmid from Escherichia coli showing changes in fluorescence were sequenced. Students used sequence alignment tools, protein translator tools, protein modeling, and visualization to analyze the potential effect of their mutations within the protein structure. This laboratory linked molecular techniques and bioinformatics to promote and expand the understanding of experimental results in an upper-level undergraduate laboratory course. Keywords: bioinformatics, GFP, site-directed mutagenesis. INTRODUCTION
This report describes a 10-week semester long experiment to utilize bioinformatics for analysis of mutations from the genetic level to the 3D protein level. The teaching goals of this laboratory were (a) to present a driving question for students to investigate and design experiments; (b) discuss problems that arise during experimentation to allow opportunities for students to apply concepts and information to solve those problems; (c) allow collaboration among students and the instructor; and (d) use of technological tools to promote inquiry. The laboratory experience presented here combined bioinformatic tools and databases with frequently used scientific techniques to mimic a research approach to a question. The experimental objective of the lab is to change GFP to a specific color chosen by the students. GFP, from the jellyfish Aequorea victoria, was discovered in the 1960s [1]. Since then, GFP has been cloned and expressed in living organisms [2]. The 3D structure of GFP was elucidated in 1996, which enabled scientists to target specific regions of GFP to genetically modify and create an array of color variants that are currently used in various biological applications [3–5]. Since its discovery, GFP has changed the way scientists visualize biological processes considerably and, in 2008, the Nobel Prize in Chemistry was awarded to Osamu Shimomua, Martin Chalfie and
Roger Tsien for ‘‘for the discovery and development of the green fluorescent protein, GFP’’ [6]. We employed both random and site-directed mutagenesis techniques to mutate GFP. To strategically mutate a protein to a desired outcome, it is necessary to analyze the nucleotide sequence, the amino acid sequence and the intramolecular interactions in the protein structure. GenBank (www.ncbi.nlm.nih.gov/genbank) and the Protein Data Bank (www.pdb.org) contain archives of nucleotide and amino acid sequences and protein structures, respectively, determined by researchers. GFP was used as the model protein for mutagenesis due to access to its nucleotide sequence, availability of its 3D protein structure, and its fluorescent capabilities, which allowed students to quickly observe any functional changes without performing additional assays. GFP has been used previously as an education tool in laboratories to visualize protein expression followed by protein purification experiments [7]. Previous educationbased articles have also used random and site-directed mutagenesis to manipulate the fluorescent properties of GFP [8–10]. Previous papers have also introduced bioinformatic tools to analyze DNA sequences or protein structure [8, 10]. In this report, the power of bioinformatics was integrated with multi site-directed mutagenesis and random mutagenesis techniques to provide students with a comprehensive demonstration of the mutational effects from the gene to the protein level.
& s
Additional Supporting Information may be found in the online version of this article. ‡ To whom correspondence should be addressed Carey L. Medin. E-mail:
[email protected]. DOI 10.1002/bmb.20554
Course Overview, Content, and Methodology Molecular Biology is an upper level biology class taken by biology majors during their junior or senior year. Bio-
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This paper is available on line at http://www.bambed.org
449 logical Principles I, Biological Principles II, Cell Biology, and Genetics are prerequisites for the course. Students must enroll in lecture and a laboratory that meets once a week for three hours. The class that performed the laboratory experience presented here was composed of 4 juniors and 13 seniors. Although the instructor defined the sequence of experiments the students would follow for protein mutagenesis, the students determined the mutation(s) that would be engineered to produce the color variant of their choice. The class was divided into four color groups with two pairs of students per color; one group contained three students. Throughout the semester students were asked to keep the overall goal in mind while understanding how each experiment helped attain the goal. Students obtained their own scientific data and used multiple bioinformatic tools for analysis of the data to determine their success at protein mutagenesis. In addition, assessment of the class, the groups and the individuals were done throughout the semester using various assessment tools (quizzes, assignments, and final paper). LABORATORY SESSIONS
Equipment The laboratory is equipped to handle up to 16 students in a given session. Larger equipment includes: a shaker/ incubator, isotemp incubator, water bath, ultraviolet (UV)– visible spectrophotometer, a UV light source, an autoclave, electrophoresis equipment, refrigerated benchtop centrifuge for 1.5 ml microcentrifuge tubes, gel documentation system, thermocycler, computer for each student, and a camera. Reagents and supplies were purchased from several companies and are referenced in the Course Schedule.
Course Schedule The following experiments were performed in sequence over 10 weeks in three-hour blocks (Table I).
Session 1-Tutorial of Protein Database and GFP and Identification of Mutations For GFP Variants After an initial lecture outlining the goals of the course, the students were introduced to the structure of GFP by using a tutorial by Milton et al. for using Protein Data Bank (PDB; www.pdb.org) [11]. The two goals of this exercise were to have students become familiar with the Protein Data Bank and the ability to visualize the overall beta barrel structure of the green fluorescent protein and the location of the chromophore region at the 3D level. Students also analyzed the effects of mutations on the structure using the 3D model. After analysis of the GFP protein structure, students initiated a literature search online using available resources to familiarize students with literature on the topic and identify mutations that change green fluorescent protein to a color of their choosing. A classroom outfitted with computers was used to allow students to perform online literature searches using databases such as ScienceDirect
TABLE I Site-directed mutagenesis experimental timeline Lab session # 1 2 3 4 5 6 7 8 9 10
Experiment Tutorial of protein database and GFP Identify mutation(s)–library Prepare media for transformations Design and order site-directed mutagenesis primers Isolate and quantitate plasmid DNA (pGFPuv) Site-directed mutagenesis using PCR DpnI digest Transformation/ random mutagenesis Screen mutants and start cultures Isolate and quantitate DNA Design sequencing primers and order Prepare samples for sequencing Review chromatograms, Go over student sequencing data – library
(www.sciencedirect.com) and PubMed (www.ncbi. nlm.nih.gov/pubmed) as part of the laboratory exercise. The students formed four color groups (orange, cyan, blue and yellow) with each color change requiring less than five amino acid changes based on the literature search. An assignment was associated with this exercise. Students were asked to use the Protein Data Bank tutorial to predict what effect their chosen mutation(s) would have on the structure of GFP. Task 1: Students used the coding sequence of pGFPuv (obtained from clontech. com and NCBI Accession: U62636) and manually entered the desired mutation(s) into the sequence. Task 2: Students entered their DNA sequence into the ExPASy website (www.expasy.org/tools/dna.html) to translate the DNA sequence into protein. In order for this to be done properly, the students must select the correct translational reading frame for producing a functional protein. Task 3: Students entered their translated sequence into the Protein Data Bank and searched the database to find a 3-D structure with the same or similar mutations. Task 4: Students discussed the potential effect their mutations will have on the beta barrel structure and the chromophore region due to the location of their mutations. Specifically, the students had to identify and describe what amino acid change they made, where in the structure the mutation was located and how the mutation could alter the chromophore color.
Session 2-Prepare Media for Transformations and Primer Design for Site- Directed Mutagenesis As preparation for the transformations performed during session 5, the students made media. Each of the seven student teams made 20 test tubes of 5 mL Luria Broth (LB) broth þ ampicillin (100 lg/mL) and 25 LB þ ampicillin (100 lg/mL) agar plates. In preparation for performing multi site-directed PCR, students used the GFP sequence (obtained from clontech.com and NCBI Accession: U62636) and followed instructions from QuickChange Lightning Multi SiteDirected Mutagenesis Kit from Agilent Technologies to engineer their desired mutations in PCR primers (Agilent Technologies, CA). Though there were four color groups, two pairs of students within the same color group often
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decided on different or additional mutations to obtain the same color. Therefore, there were seven teams that used different primers to achieve their desired GFP variant. Many of the GFP variants required more than one mutation and as a result, multiple primers were used to anneal to adjacent or well-separated regions on the same strand of DNA (see Table IV in supplemental information). Once the students finalized their primer sequence, they entered their primer sequence into an electronic document to order from Integrated DNA Technologies (IDTDNA; idtdna.com).
hemi-methylated. Therefore, unmethylated PCR products that contain the mutation(s) remained after digestion. Students ran 10 lL of the DpnI digest on a 1% agarose gel containing 1 lg/mL ethidium bromide and used a gel documentation system to verify the presence of a PCR product. Because of ethidium bromide’s carcinogenic effects, students were required to wear gloves while adding ethidium bromide to gel and handling and analyzing the agarose gel. The agarose gel, gloves, and tips containing ethidium bromide were disposed of in a separate biohazard container.
Session 3-Isolate and Quantitate Plasmid DNA (pGFPuv) and Site-Directed Mutagenesis of GFP
Session 5- Site Directed Mutagenesis and Random Mutagenesis Transformations
As preparation for the site-directed mutagenesis of GFP, each student team had to isolate plasmid DNA containing the GFP gene (pGFPuv from Clontech) from a stock bacterial culture. Prior to the session, the instructor transformed chemically competent E. coli with isolated plasmid DNA containing the GFP gene (pGFPuv) from Clontech. The day before session 3, the instructor made an overnight 100 mL bacterial culture in LBþ ampicillin (100 ng/mL). The students took aliquots of this culture and performed a plasmid miniprep using the Qiagen miniprep kit (Qiagen, CA) to isolate pGFPuv followed the instructions provided by the manufacturer. After isolation, the students determined the concentration of purified plasmid DNA using a UV/Vis spectrophotometer. DNA concentration was measured using the absorbance at 260 nm. Next the students diluted their site-directed primers obtained from IDTDNA to a final concentration of 100 ng/lL in dH2O. The students set-up and performed sitedirected mutagenesis following the instructions included in the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). The kit is designed to incorporate mutations at multiple sites in the dsDNA in one PCR reaction based on the number of primers (containing individual mutations) that are added to the reaction. Each site-directed PCR reaction consisted of 50 ng of pGFPuv that the students isolated, 1 lL of QuikChange Lightning Multi enzyme blend, 2.5 lL of 10X reaction buffer, 1 lL of dNTP mix, 100 ng of each primer for 1–3 primers containing separate mutations) or 50 ng for each primer for 4–5 primers (containing separate mutations). Cycling conditions consisted of 958C for 2 min followed by 30 cycles of: 958C for 20 sec, 558C for 30 sec, and 658C for 2 min and an extension cycle at 658C for 5 min. Since the cycling took over 3 hr to complete, the instructor removed the PCR reactions when the thermocycler finished cycling and stored them at 48C until the next laboratory session.
The students transformed chemically competent E. coli (XL10-Gold Ultracompetent cells, Stratagene, Agilent Technologies) provided by the site-directed mutagenesis kit with their mutated plasmid DNA. They also performed a transformation with unmutated pGFPuv as a positive control for GFP expression. The following adjustment was made to the manufacturer’s transformation protocol; the transformed cells recovered in LB broth made by students during Session 2 instead of NZYþ broth. Just prior to plating, the students added 100 mM Isopropyl-betathio galactopyranoside (IPTG) to the LB þ ampicillin (100 ng/mL) plates that they made during Session 2. GFP expression is controlled by the lac operon in the plasmid pGFPuv. Therefore, the lactose analog IPTG was added to induce expression of GFP. The transformed cells were plated on these plates, incubated at 378C overnight and stored at 48C until the next laboratory session. In addition to multi site-directed mutagenesis, students transformed the XL1-Red E. coli with pGFPuv plasmid. XL1-Red E. coli are deficient in three of the primary DNA repair pathways. The mutS (error-prone mismatch repair) mutD (deficient in 30 - to 50 -exonuclease of DNA polymerase III) and mutT (unable to hydrolyze 8-oxodGTP) mutations allow a random mutation rate to be 5,000-fold higher than that of wild type [12–14]. The random mutagenesis was used as an alternative approach to mutagenizing the GFP gene. Students transformed XL-1-Red competent cells with 50 ng of pGFPuv plasmid the following the procedures from the manufacturer (Stratagene). Transformed cells recovered in LB broth and were plated on LB-amp (100 lg/mL) 2IPTG (100 mM) plates. The plates were incubated at 378C for 48 hours and stored at 48C.
Session 4-DpnI Digest of PCR Products The students digested their PCR product by adding 1 lL of DpnI (supplied in the multi site-directed mutatgenesis kit) directly to their PCR reactions and incubating at 378C for 5 min. DpnI digests DNA that is methylated or
Session 6-Screen for GFP Color Variants Colonies that grew after transformation of E. coli with plasmids from multi site-directed and random mutagenesis experiments were compared to E. coli transformed with wild type pGFPuv using a UV light source. The students assessed colonies for any color changes when compared to the wild-type GFP on positive control plates. Up to three colonies from site-directed mutagenesis experiment and two colonies from the random mutagenesis experiments were selected for further analysis.
451 TABLE II Sequencing primers Primer
Query: GFP gene sequence (pGFPuv nt 289 – 1005)
Sequence
GFP sequence Forward Primer
5- TTA CCT GTC GAC ACA ATC TGC CCT 23
GFP sequence Forward Primer
5- CAA GAC GCGTGC TGA AGT CAA GTT 23
GFP sequence Reverse Primer
5- TCC ATG CCATGT GTA ATC CCA GCA 23
GFP sequence Reverse Primer
5- TGA CAA GTGTTG GCC ATG GAA CAG 23
The query is the GFP sequence from GenBank Accession #U62636. The subject is the primer. Sequences were aligned using blastn (NCBI).
The criteria for selection of colonies depended on whether the fluorescence under UV light appeared brighter, dimmer, knocked out or a different color altogether compared with wild-type fluorescence. The students streaked the chosen colonies on an LB/ampicillin/ IPTG plate and incubated them overnight at 378C and stored at 48C until the students were ready to start overnight cultures for plasmid isolation.
Boston, MA (tufts.org). The students set up their sequencing reactions. Each sequencing reaction contained 0.5 lg/lL of plasmid DNA and 25 ng/lL of each primer (reverse and forward). Students entered their own sequencing reactions information online to submit tufts.org. The tubes containing the primers and plasmid DNA were shipped to Tufts Core Facility for sequencing.
Session 10-DNA Sequencing Analysis Session 7-Isolation of Mutated Plasmid DNA The day before the laboratory Session 7, students observed their streak plates under UV light and looked for phenotypic changes. The students chose colonies that contained phenotypic changes to inoculate 5 mL bacterial cultures in LB/ampicillin broth. The bacterial cultures were incubated at 378C overnight in a shaking incubator. During laboratory Session 7, the students isolated plasmid DNA from their bacterial cultures using the Qiagen miniprep kit following the instructions provided by the manufacturer. After isolation, the students determined the concentration of purified plasmid DNA and measured DNA concentration as described in Session 3.
Session 8-Designing Sequencing Primers In preparation for sequencing mutated plasmids, a lecture on sequencing and the most common factors that influence the acquisition of good sequencing data was presented. As a class, the students used the formatted GFP sequence from the pGFPuv plasmid and designed primers for sequencing using PrimerQuest software (idtdna.com). Four primers were chosen based on the criteria of sequencing overlapping regions of the GFP gene and were spaced 500 bp apart (Table II). The instructor ordered the primers online from IDTDNA.
In preparation for analysis of sequencing data, students were shown example chromatograms and sequence alignments to identify pitfalls and potential problems with sequencing data. This includes height and breadth of peaks, incorporation of N nucleotides, background noise (appearance of baseline peaks) and undesirable data at the end or beginning of a sequence reaction. After the examples were given, the students were given a three part assignment to refine their own sequencing data and determine whether the proper mutations were incorporated for their site-directed mutagenesis and their random mutagenesis. The first part of the assignment was to identify ‘good’ sequence data by analyzing the peaks associated with each nucleotide in the chromatograms. If possible, the students verified any DNA sequence that was not ideal with a sequence from an overlapping region obtained from a sequencing reaction using a different primer. The second part of the assignment required students to copy the sequence associated with the chromatograms and align the sequence with the unmutated GFP sequence using Blast software from NCBI (blast.ncbi.nlm.nih.gov/Blast.cgi). The third part of the assignment required students to write a summary of their sequencing results. In addition, the students discussed how the sequence they obtained correlated with a change in the fluorescence (i.e. change of color, knock down, knock up). STUDENT RESULTS
Session 9-Preparation for DNA Sequencing Once sequencing primers were obtained from IDTDNA, the students diluted them with dH2O to a concentration of 5 lM (25 ng/lL) as directed by Tufts Core Facility,
Of the seven student teams, three observed phenotypic changes in GFP. Two of the three teams visually saw a color change in their transformed E. coli while the third group saw a knockdown in GFP fluorescence
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FIG. 1. Change of fluorescence after site-directed mutagenesis. Bacterial cells were transformed with pGFPuv plasmids containing mutations within the GFP gene. Colonies were restreaked on Luria agar plates containing 100 lg/ml ampicillin and 100 mM IPTG and grown overnight at 378C. (a) Colonies were visualized under UV light to assess color changes. (b) Colonies were visualized under UV light to assess colony growth and color changes. Colonies containing the following plasmids in clockwise direction starting from the top right: 1. colonies containing Y1 plasmid from Group 1; 2. colonies containing Y2 plasmid from Group 1; 3. colonies containing GFP plasmid (pGFPuv); 4. colonies containing Y3 plasmid from Group 2; 5. colonies containing Y4 plasmid from Group 2, 6. colonies containing GFP plasmid (pGFPuv); and 7. colonies containing C1 plasmid from Group 3.
(Fig. 1). The goal of the two groups that saw a color change was to mutate GFP to YFP. Group one designed their primers to incorporate amino acid changes S65G, V68L, Q69K, and T203Y (Fig. 2). Two colonies were chosen from their site-directed mutagenesis plate for further analysis, one with a change in color of fluorescence, Y1, and one with a knockdown in fluorescence phenotype, Y2, (Fig. 1a-1 and 22, respectively). Y1 had only one intended amino acid change, the S65G change, incorporated. The serine to glycine change at amino acid position 65 in Y1 created a shift in the fluorescence from green to yellow-green when compared with wild type GFP (Fig. 1a-3 and 26, respectively). Interestingly, E. coli transformed with Y1 plasmid were yellow in white light (Fig. 1b-1) and fluoresced more intensely than any of the other colonies. Y2 also had only one intended amino acid change, the S65G change, incorporated. However when the students analyzed the sequencing data, they found an additional mutation in Y2. Y2 had a deletion at nucleotide 909 causing a frame shift downstream. This
mutation was most likely the cause of the knockdown phenotype seen for Y2 (Fig. 1a-2). Group 2 was the second team to attempt to change GFP to YFP. The mutations this group intended to incorporate by site directed mutagenesis were S65G, V68L, Q69M, S72A, and T203Y (Fig. 2). Two transformed E. coli colonies were chosen from the site-directed mutagenesis plate based on different intensities of fluorescence. Y3 fluoresced yellow-green and Y4 was a dim yellow color (Fig. 1a-4 and 25, respectively). Y4 variant contained three mutations that were incorporated into the GFP gene, the S65G located in the chromophore of the protein, the S72A mutation and the T203Y (Fig. 2). The Y3 variant also had mutations incorporated, but only the S65G and T203Y mutations, which could explain why Y3 was yellow-green rather than yellow. The goal of Group 3 was to incorporate the mutations F64L, S65T, Y66W and N149I to create the color cyan (Fig. 2). Unfortunately, the colonies on their site directed mutagenesis plate had a knockdown in fluorescence
FIG. 2. Review amino acids successfully mutated within GFP by students. The arrows indicate regions within the GFP protein that were targeted for site-directed mutagenesis. The amino acids in red for each group reflect successful incorporation of the mutation. Illustration was created using Protein Workshop at PDB.org.
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FIG. 3. Group 3 alignment of primer with GFP sequence. Subject is the primer designed by Group 3 to incorporate the nucleotide changes resulting in amino acid changes F64L, S65T and Y66W. The Query is the GFP sequence from GenBank Accession #U62636. Both sequences were aligned using blastn (NCBI). The regions that are unmatched between query and subject indicated engineered changes in the nucleotide sequence. The nucleotides indicated by a red box were errors incorporated into the primer design. At position 504, a thymine (T) replaced a cytosine (C), whereas, a deletion of a thymine (T) was incorporated. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
phenotype when compared with wild type (Fig. 1a-7 and 26, respectively). After sequence analysis of their plasmid DNA, C1, the group found that all the intended mutations were incorporated. The results contradicted what was found from their literature search. However, after sequence analysis, the students discovered that they had made an error in their primer design. The students had designed a primer with a T instead of a C at nucleotide position 504 and left out a T at position 507 causing a frame shift mutation downstream (Fig. 3). The error in primer design was most likely the reason for the knockdown phenotype instead of a change in color. This experiment emphasized to the students the importance of each nucleotide in producing a functional protein. The other four groups were unsuccessful in obtaining GFP variants. Although the success rate was low at achieving the final goal of changing GFP fluorescence, students were successful at many steps of the laboratory exercise such as obtaining PCR product, transformations, and sequencing analysis. When plasmid DNA was isolated from colonies on their site-directed mutagenesis plates and sequenced, the sequence data either indicated that the groups did not obtain their intended mutations or the quality of the chromatograms was too poor to conclude whether they had incorporated their mutations. Some groups had a lot of background noise in their chromatograms that made it difficult to distinguish sequences. It is possible that the background noise could have been due to a failed or incomplete DpnI digest, which would result in a combination of parental and nonparental DNA. Background noise could also have been observed if the students did not obtain a single colony for DNA isolation and therefore, the plasmid DNA sent out for sequencing contained two different plasmids. In addition to background noise, some sequencing data was missing large regions of nucleotides, indicating that the sequencing reaction was not optimal. It was unclear as to why some sequencing reactions worked well and others did not. However sequencing reactions require students to pipet very small volumes. Any error is student pipetting could lead to failure of the sequencing reaction.
Evaluation of Students The students’ grades for the laboratory course were determined by their graded work; a laboratory notebook (20%), lab quizzes (25%), three written assignments (Assignment #1: bioinformatics (see Session 1), Assignment #2: cloning problem, and Assignment #3: sequence analysis [see Session #10)] (25%), and a final paper (30%).
As scientists, it is imperative that scientists keep an accurate notebook of their experiments. Therefore student notebooks were maintained over the semester and an assessment of their laboratory notebook was done to ensure the notebooks contained the necessary information to repeat the experiments and the summary of the final results. To ensure that individual students understood the sequence, design, and purpose of experiments, students were given occasional quizzes and written assignments. Questions on the quizzes required students to explain the experiment that they had performed the previous laboratory session. The three written assignments assessed the students’ abilities to comprehend the overall concepts of the lab such as primer design, cloning, purpose of reagents used, and so forth. The assignments required students to use the skills that they acquired to either design or explain the experiment that they had performed or a similar experiment. The final paper was written following the guidelines from the Journal of Molecular Biology or the Molecular and Cellular Biology Journal. The article required students to incorporate the assignments for their literature search to identify the mutation(s) required to make their particular color (see Session 1), how their intended mutations would affect the GFP protein (see assignment for Session 1) and the sequence analysis of their clones (see assignment for Session 10).
Assessment of the Laboratory Experience The laboratory experience presented was designed to achieve four main goals described in the Introduction. At the start of the semester, students were asked to identify mutations that will change the color fluorescence of GFP and design experiments to implement the mutations to GFP (first goal). Discussion and development of the experimental design helped the students apply the information they learned in their classes. The impact that the laboratory experience had on student learning was reflected in the online Student Assessment of their Learning Gains (SALG) survey (www.salgsite.org) that students filled out at midterm and at the end of the semester. In the SALG survey, several students reflected that the designing aspect of the experiments increased their understanding and retention of molecular biology. One student commented that ‘‘The concepts of designing primers to make the specific mutation desired is much more clear having done it ourselves. Also I understand the process of cloning and what each step actually does much better.’’ Another student commented that ‘‘The biggest contributor to retaining this information has
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come from actually doing it ourselves. Ideas are clarified exponentially when you actually sit down and do it in lab.’’ In addition to designing each aspect of the experiments, students had to follow manufacturers’ instructions from plasmid isolation kits, multi site-directed mutagenesis kits, and primers. They used reagents straight out of the boxes instead of having them premeasured and aliquoted for them. Reagents not supplied by kits had to be prepared by the students. This helped the students understand the complexities involved in scientific experiments that they were not previously exposed to. It emphasized how much effort goes into answering a scientific question and that each scientific article is a culmination of months and, many times, years of research. As a result, students developed a sense of ownership of their work in the lab. One student commented in the SALG survey that ‘‘I liked the fact that the students did most of the lab on their own. I found it much more engaging and that the responsibility of doing these things made everything more real as well as giving it a higher priority.’’ When a problem arose, the class finished what they were doing and then as a class, brainstormed and hypothesized the results of the error (second goal). Next the class assessed whether they could continue their experiment with this error and brainstormed solutions to mediate the error. These sessions allowed students to think about what was happening each step of the way. The students determined whether the errors were critical and the experiment needed to be redone or if not, what the results may look like. For example, one group put their DpnI digest into a 908C water bath instead of 378C degree water bath. The instructor brought the class together to assess the situation. The class determined that if the group continued forward, they might have a mix of parental and nonparental DNA when they plated their transformed cells. Under the guidance of the instructor, the class and the group decided that this would not harm their experiment so they continued forward with their experiment. When the students analyzed their sequencing data, they revisited this experimental error.
They hypothesized that a failed DpnI digest could have lead to isolation of a mixture of wild type and mutated DNA, which could have given rise to the poor quality of sequencing data obtained. The group gained valuable insight into the importance of each step in an experiment. Another example was when a group forgot to add LB broth for bacterial recovery after transformation. The class came together and decided that the cells would not survive in the harsh environment of the transformation solution and the group redid the transformation. These sessions were important in emphasizing what was happening at each step of the process. In addition, it showed students that science experiments have some flexibility in some cases but not in others. This was a novel approach to thinking in the lab for the students. One student commented in the SALG survey that ‘‘It is much more similar to working in a real lab and I think you learn more by figuring things out yourself rather than having a teacher figure them out for you and then tell you what to do.’’ Another student commented that ‘‘I liked being able to talk to my group so that we could discuss what was going on, what needed to be done, and brainstorming how we could fix problems.’’ The laboratory experience fostered regular collaboration among students and the instructor (third goal). Students collaborated with one another to choose the mutations, to design primers to produce the mutations, to do the experiments in the lab, to interpret and analyze results from each experiment, to troubleshoot problems in the lab, and to help each other understand the overall project and the experiments involved. The instructor served as a guide, providing pre-lab lecture information, and fostering discussions in the lab and outside of the lab. The importance of collaboration was evident in the SALG survey where students reported that they made gains at working effectively with others (midterm percentage ¼ 68.8%, final percentage ¼ 72.7%) and that the lab impacted their willingness to seek help from others when working on academic problems (midterm percentage ¼ 62.5%, final percentage ¼ 72.7%) (Table III). One student commented in the SALG survey that ‘‘I think working
TABLE III Results of student assessment of learning goals (SALG)
How much did each of the following help in your learning? Assignment 1: Bioinformatics Assignment 2: Cloning problem set Assignment 3: Sequence Analysis Final Paper Graded assignments (overall) What gains did you make in the following skills? Working effectively with others How did the lab impact your attitudes? Confidence that you understand the material Confidence that you can do this subject area Your comfort level in working with complex ideas Willingness to seek help from others (teacher, peers) when working on academic problems
Percentage of responses that were 3 s. 4 s or 5 s (N ¼ 16 unless indicated otherwise)
Percentage of responses that were 3s. 4s or 5s (N ¼ 11 unless indicated otherwise)
64.3% (N ¼ 14) N/A N/A N/A 64.3% (N ¼ 14)
81.8% 81.8% 90.9% 66.7% (N ¼ 9) 72.7%
68.8%
72.7%
93.8% 93.8% 93.8% 62.5%
81.8% 81.8% 81.8% 72.7%
The following scale was used for assessment; 1 ¼ no help/gain/impact, 2 ¼ a little help/gain/impact, 3 ¼ moderate help/gain/impact, 4 ¼ good help/gain/impact, and 5 ¼ great help/gain/impact.
455 with others is the most helpful in my learning because we can talk things out and learn from each other and help each other too.’’ Another student commented that ‘‘Working in a group was helpful in learning because we were able to discuss what was being done, why, and explanations of the results.’’ The results from the SALG survey in Table III indicate that the emphasis on including bioinformatics in the laboratory curriculum helped students learn (midterm percentage ¼ 64.3%, final percentage ¼ 81.8%) (fourth goal). The use of bioinformatics analysis was crucial in allowing the students to relate protein function with nucleotide sequence. Students were able to observe a change in the color of fluorescence and then correlate it with the incorporation of their mutations after they completed the sequence analysis. Furthermore, they could see the position of the mutation(s) in the 3D protein model using the Protein Data Bank (pdb.org). Students were also able to observe a knockdown phenotype in the lab and detect frame shift mutations when they performed their sequence analysis. One student group used sequencing alignments to review their primer design from the initial experiments and identified errors in primer design that lead to the frame shift mutations. The use of bioinformatics to align DNA sequences and position mutations in the protein model along with observation of phenotypic changes emphasized how important each nucleotide is in producing a functional protein. This is a concept that has been presented to the students in their freshman and sophomore courses but they have never seen for themselves until this lab taken in their junior or senior year. Students were excited to observe this concept in the lab. One student commented in the SALG survey that ‘‘I found the visual aids in the PDB website to make the class more interesting, and easier to understand what was happening with the protein.’’ The students indicated that the assignments were important for the laboratory experience and using bioinformatics tools (Table III). The assignments guided students through the processes of using GenBank to find and retrieve DNA sequences so that they could design primers, using the Protein Data Base to observe predicted protein structure and determine which regions would be mutated and using BLAST software to align DNA sequences. The students viewed the assignments as tools to help them process and comprehend their experimental results. They also felt that using bioinformatics in the class together and in guided assignments made it less intimidating to use technological tools that were novel to them. The assignments provided them with opportunities
to explore their results on their own and receive direct feedback from the instructor. There were many positive student comments about the assignments in the SALG survey. One comment that exemplifies students’ attitudes towards the assignments is ‘‘The second part of the first assignment was really helpful in further understanding and analyzing what my specific mutations might do to GFP and allowed me to reflect on why we made the mutations we did and explore those reasons further.’’ Another student commented that ‘‘All of the homework assignments really helped to organize what we had done up until that point, and figure out the logic and reasoning behind the experiments. I really thought the homework assignments were extremely helpful.’’ A novel outcome of the laboratory experience was observed. Students indicated that they had more confidence in themselves. The SALG survey indicated that the students felt more confident in understanding (midterm percentage ¼ 93.8% and final percentage ¼ 81.8%; Table III) and working with complex ideas (midterm percentages ¼ 93.8% and final percentage ¼ 81.8%; Table III). One student commented in the SALG survey that ‘‘The class hasn’t changed how I study, but it has helped me gain confidence when working with a lot of information to try and piece together complex ideas.’’ The students also indicated that they were confident that they could do this subject area (midterm percentage ¼ 93.8% and final percentages ¼ 81.8%; Table III). One student commented in the SALG survey that ‘‘since the experimentation done in this class is mostly independent, I feel more confident that I can do research-oriented work on my own, whereas before I was more nervous about proceeding in lab without explicit instructions.’’ However, one student did not feel as comfortable with the guided instructional approach as demonstrated by the comment ‘‘Explanations helped, but I felt like lab should have involved slightly more instruction.’’ Overall, both the students who obtained a color variant and those that were unsuccessful felt they gained greater confidence in applying bioinformatic tools to scientific experiments in molecular biology by taking this laboratory exercise. An important factor to consider with a lab curriculum such as this is the cost of reagents and kits. Table IV lists the major costs associated with this laboratory 10-week sequence with 17 students (eight groups). The table does not include common materials (such as media, dNTP’s, agarose) or equipment (such as a PCR thermocycler, incubator, gel boxes, or heat block). This 10-week semester-long project can be performed by 17 students for $2400 ($141/student). Reagents purchased the first
TABLE IV Cost analysis of 10-week laboratory Reagent Mutagenic primers (HPLC purified) Multi site-directed mutagenesis kit Miniprep kit XL-1 Red Competent Cells Sequencing primers Sequencing reactions (Tufts) Total
Approximate cost $55/primer (18 primers) $330 for 10 reactions $80 for 50 samples $380 $12/primer (4 primers) $9/reaction (up to 8 per group)
Total cost (17 students) $990 $330 $80 $380 $48 $576 $2404
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time running the laboratory exercise can be used in the following semesters such as primers and unused portions of kits. FUTURE IMPROVEMENTS
One consideration for improving the laboratory exercise was allotting enough time for the students to design and review their primers. There was one laboratory session set aside for primer design. In light of the errors that were discovered after sequencing, it is clear that the students may have benefited from having an assignment that asked students to BLAST their primers against the wild type GFP gene. This would have ensured that they incorporated the intended mutations without causing unintended frame shift mutations. The second key improvement would be to have a set of primers and a plasmid that contained mutation(s) already experimentally done by the instructor or ones produced by previous students to be used as a positive control during the experimental process. Although the students who were unsuccessful in obtaining a color variant did not have a negative comment on the lack of results, a positive control to verify that the students were performing the experiments correctly would be helpful. Additionally, a caveat of ordering primers and sending plasmids out for sequencing means that enough turnaround time needs to be scheduled. Typically, one week is sufficient; however, synthesis of primers can be delayed if synthesis of primers fails initially and sequencing of primers could be delayed by any number of reasons. Our class experienced this type of delay when one group’s set of site-directed mutagenesis primers took longer to synthesize than the rest of the class. Fortunately, the group was able to come in on their own time to perform PCR so the group did not fall behind schedule with the rest of the class. To avoid any delays, it would be advisable to include a flex lab day for any issues that might arise. Another time saver would be for the instructor to prepare media, solutions, isolate the initial pGFPuv DNA, and aliquot reagents to accommodate shorter lab sessions or semester schedule.
Acknowledgment— The authors would like to thank the students who took the Molecular Biology lab in the fall of 2011. They would also like to thank Sharon Ramos-Goyette, Bob and Diane Peabody, John Lanci, Stacy Grooters, Bronwyn Bleakley and Craig Almeida for their help and advice during the writing process. They would also like to thank Dr. Alan Rothman for his valuable critique of the manuscript.
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