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Keywords:

  • Undergraduate;
  • plasmid;
  • cloning;
  • project-based laboratory

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the classroom because of the preparation, expense, and logistics involved in independent student projects. The authors have designed a 10-week lab series that mimics the research environment by tying separate fundamental lab techniques to a common goal: to build a plasmid with yeast actin cDNA cloned in a particular orientation. In the process of completing this goal, a problem arises in that students are unable to obtain the target plasmid and instead only recover the gene cloned in the opposite orientation. To address this problem, students identify four plausible hypotheses and work in teams to address them by designing and executing experiments. This project reinforces the utility and flexibility of techniques covered earlier in the class and serves to develop their skills in experimental design and analysis. As the project is focused on one problem, the diversity of experimental approaches is limited and may be prepared in advance with little additional expense in reagents or technical support.

As research in virtually every field associated with the life sciences has drifted toward the use of molecular techniques, laboratory courses in molecular biology have become a vital part of the undergraduate curriculum. Although there is a significant accumulation of literature emphasizing the importance of the development of critical thinking skills [1, 2], they are often ignored in these courses because of expense, logistics, and technical support. These courses have been designed with the primary focus on training the students to become proficient in techniques, but many have not emphasized how one is to select the techniques and proper controls to address a novel problem. The end result is that our students enter the research environment in academia or industry without having a clear idea as to how to apply the techniques they have learned. As many of the students who take our course are in pursuit of graduate programs and local/regional biotech industry (Baxter, Amgen, Stratagene, Promega, Santa Cruz Biotech, etc.), this emphasis on application is particularly important for our degree program.

So, how does one train students in the correct molecular techniques while simultaneously encouraging them to develop their critical thinking skills in a manner that is feasible for an undergraduate course? In our molecular biology laboratory course, we have followed a skills matrix that has been described for such courses [3] to design a laboratory series that exposes students to an environment that is similar to what would be experienced in a research laboratory. On the first day of class, a project goal is presented to them: to construct a plasmid that contains the yeast actin cDNA cloned into the lacZ′ locus such that it could be expressed using the lacZ′ promoter (i.e. cloned in the “sense” orientation). As they conduct experiments aimed toward achieving their goal, they develop skills in core molecular techniques (see flow chart in Fig. 1). To better simulate the realities of scientific research (and to stimulate critical thinking), the lab series also includes a problem: none of the bacterial transformants that were expected to harbor recombinant plasmids carried plasmids with the desired insert orientation (pACT-S, see Fig. 2). It has been demonstrated that experience with “real life” research problems, involving student participation in experimental design and data analysis/interpretation, enhances student learning [4]. To identify the cause of this problem, our students are required to use their background knowledge and understanding of molecular techniques to propose and test hypotheses that would account for their inability in obtaining the “correct” plasmid. By repeating many of the same techniques that were learned earlier in the quarter (e.g. PCR, ligation, transformation, plasmid isolation, restriction mapping), this approach has the added benefit of repetition, which is known to improve the proficiency in the techniques and illustrates how they may be utilized (and even manipulated) to address a novel problem [5].

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Figure 1. Class flow chart of the experimental design used in the Cal Poly Molecular Biology laboratory course. The shaded regions and thick lines represent the procedures followed and the desired products from each step (the goal for this project is represented in the black box at the bottom left). The rejected products from the RT-PCR, alpha-complementation, and restriction mapping screens are represented in the white boxes with thin lines linking them to the chart.

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Figure 2. Class restriction mapping results illustrate unexpected orientation bias. (a) Graphic representation of the two expected recombinant plasmids following the nondirectional ligation reaction. (b) Representative class data from a HindIII digest of plasmids derived from white colonies. Lane 1 corresponds to the 1-Kb DNA ladder (Promega) and lanes 2–8 are digestion products of isolated plasmids. Only the pACT-A plasmids (carrying the anti-sense orientation of ACT1) are represented, as shown by the digestion products of 4.6-Kb and 425-bp bands. The faint band below 4.6 Kb represents incompletely digested plasmid. If the pACT-S plasmid had been present, bands would have appeared at 4.2 Kb and 880 bp.

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METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

PCR-based Procedures

Site-specific PCR mutagenesis was performed to mutate the putative -10 and -35 sigma factor binding sites found on the anti-sense strand of SpACT1 cDNA. The SigMut-F (5′-CGG TAA CAT CGT Tat gag tGG TGG TAC CAC CAT G-3′) and SigMut-R (5′-TAA CGA TGT TAC CGt aca gcT CCT TAC GGA CAT CG-3′) primers were designed to mutate the binding site (shown in lower case) while preserving the translated sequence of actin. The ACT1 cDNA was amplified in two separate reactions: 1) 900 bp of the 5′ region using ACT-F and SigMut-R primers and 2) 300 bp of the 3′ region using SigMut-F and ACT-R primers. The products were cut out of an agarose gel and purified (UltraClean GelSpin kit, Mo Bio Labs, Carlsbad, CA). An overlap of 14 bp between the two fragments (5′ ends of SigMut primers) was elongated to generate a more effective PCR template by denaturing the DNA fragments (95 °C for 2 minutes) and combining 1 ng of each product (or only one for the single fragment controls) with 1× Buffer B (Promega, Madison, WI), 0.8 mM dNTPs, 2 mM MgCl2, and 1 U of T4 DNA polymerase (New England Biolabs, Ipswich, MA). After a 30-minute incubation at 37 °C, the reaction was set at 95 °C for 2 minutes and then cooled to 80 °C before adding 1 μM of the ACT-F and ACT-R primers and 1 U of Taq polymerase (Promega). Amplification of the full-length (mutated) actin cDNA was performed using the following thermocycling parameters: 25 cycles of 95 °C for 0.5 minutes, 55 °C for 1 minute, and 72 °C for 1.5 minutes with a final hold at 72 °C for 5 minutes.

Colony PCR was used to amplify the multiple-cloning site (MCS) of plasmids propagated in colonies grown on LB-AX plates (LB agar supplemented with 100 μg/mL of ampicillin and 40 μg/mL of X-gal (Molecular Probes, Eugene, OR)). The reaction mix consisted of 25 μL containing 0.8 mM dNTPs, 2 mM MgCl2, 0.1 μM of each primer flanking MCS of pCR2.1 (TS315F: 5′-CCA TTC AGG CTG CGC AAC TG-3′; TS315R: 5′- CGA CTG GAA AGC GGG CAG TG-3′), and 1 U Taq DNA polymerase (Promega). Each colony was gently touched with a sterile pipette tip to obtain a sample of the cells, which were then thoroughly resuspended in the 25 μL reaction. Prior to performing PCR, a 5-μL sample of this mix was then spotted onto LB-AX to verify colony color. The reaction mix was then heated to 95 °C for 2 minutes to lyse the cells and denature the plasmids. The MCS was amplified from the plasmids by cycling through 25 rounds of 95 °C for 0.5 minutes, 55 °C for 1 minute, and 72 °C for 1.5 minutes, followed by a final elongation at 72 °C for 5 minutes. The amplified products were digested with HindIII (New England Biolabs) in a 20-μL reaction consisting of 5 μL of the amplified product, 1× Buffer 2, and 10 U of HindIII enzyme. Digested products were analyzed by agarose gel electrophoresis.

Plasmid Construction and Analysis

PCR products of wild type and SigMut ACT1 cDNA were ligated into pCR2.1 and transformed into TOP10 or TOP10-F′ chemically competent cells using the protocol and reagents supplied in the TOPO-TA kit (Invitrogen, Carlsbad, CA). Transformed cells were either spread onto LB-AX plates or grown in LB broth supplemented with 100 μg/mL ampicillin and grown overnight at 37 °C. Full development of blue and light blue colonies appeared after storing plates at 4 °C overnight. Liquid cultures of transformed populations (grown directly from transformation mix) or from individual colonies were used for plasmid isolation using the UltraClean Mini Plasmid Prep kit (MoBio Laboratories). To determine the orientation of the actin insert, isolated plasmids were digested with HindIII in a 10-μL reaction volume by incubating 5 μL of plasmid with 1× buffer 2 and 10 U of HindIII restriction enzyme (New England Biolabs) for 60 minutes at 37 °C. To examine growth characteristics on induction of lacZ′, colonies from TOP10-F′ plates were replica-plated onto fresh LB-A and LB-AI (LB-A supplemented with 100 μg/mL IPTG (Sigma-Aldrich, St. Louis, MO)).

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Setting up the Problem

Our class meetings are twice a week, each being 3 hours long, for 10 weeks. This course services advanced undergraduate students who have completed courses in organic chemistry and genetics. Most of our students are majors within the departments of biology and chemistry who have concentrations that require the class or are taking it as an elective to learn molecular techniques. Because of high student demand, we offer five sections of the course (including summers), with a maximum of 20 students per section. The goal set before the class is to clone the cDNA of the ACT1 gene from S. cerevisiae into a plasmid vector in the sense orientation relative to the lacZ′ locus (see pACT-S in Fig. 2). To achieve this goal, the first 7 weeks of the quarter are spent preparing the insert, cloning it into a vector, transforming it into E.coli, isolating the plasmids, and using restriction endonucleases to determine if the correct plasmid was isolated.

Following RNA isolation and cDNA synthesis, the actin cDNA is amplified and may be discriminated from products that arise from contaminating gDNA by size: amplified cDNAs are 1.2 kb whereas those from gDNA templates bear a 300-bp intron and are thus 1.5 kb. The reactions that contain the highest yield and purity of amplified cDNA are used in the TOPO-TA cloning kit (Invitrogen) to ligate the amplified product into pCR2.1 in a nondirectional manner. These ligation products are transformed into E.coli and plated onto selective media containing X-gal. Each student then isolates plasmids from four cultures propagated from white colonies, representing clones bearing recombinant plasmids (see [6] for review of the alpha-complementation screen). The presence of the insert may also be verified by a diagnostic EcoRI digest, which cuts twice within pCR2.1 immediately flanking the T/A cloning site (see pCR2.1 map [7]).

As the amplified cDNA is cloned into the vector in a nondirectional manner, it is expected that students would recover a 1:1 ratio of plasmids with the sense or anti-sense orientation of the actin cDNA insert. Thus, students must screen for pACT-S by performing a restriction digest to discriminate between the two possible recombinant plasmids. This mapping procedure is facilitated by the fact that the plasmid vector and insert sequences are known and downloadable from the Web (for pCR2.1 sequence, see [7]; for ACT1 sequence, see [8]). A brief meeting in a computer lab allows the students to obtain the sequence of the amplified insert (using the primers to define the ends) and build the two possible recombinant plasmids “in silico” using DNA Strider or a similar DNA analysis program. Students draw out the plasmid and indicate where the “perfect enzyme” would cut so as to allow them to easily identify the orientation of the insert. Once they have determined that the enzyme would cut once in the vector near the cloning site and once in the insert, also close to one of the ends, they search for this activity among the enzymes in the program's enzyme library. HindIII (as well as KpnI) satisfy these requirements and are used by the students in the following wet lab to map their plasmids. The typical result from this analysis is that every student isolates only one of the two possible recombinant plasmids (pACT-A, see Fig. 2), which is not the plasmid needed to accomplish the project goal. To explain this unexpected result and attempt to obtain the pACT-S plasmid, a lab meeting was used for students to form small groups and, with limited guidance, discuss the problem and identify the following four possible causes of the failure:

  • 1)
    the topoisomerase in the TOPO-TA cloning kit only effectively ligates the cDNA into pCR2.1 in the anti-sense orientation;
  • 2)
    the sense orientation of the insert results in the expression of yeast actin, which is toxic to the strain of E.coli being used;
  • 3)
    the insert orientation affects the replication of the plasmid and thus the host cell is unable to effectively grow in selective media;
  • 4)
    the pACT-S plasmids are propagated in colonies that normally represent self-ligation events (blue) and are not chosen for analysis.

Addressing the Problem

To determine if one or more of these possibilities is responsible for the failed attempt to recover the plasmid of interest, they are converted into null hypotheses and the students design experimental approaches to address each. Those procedures required for the investigative portion of the class were not included in the manual and only provided 2–3 days in advance of the lab activity to promote student participation in predicting the steps necessary for the project. Students worked in teams of four to address each of the hypotheses during the last 3 weeks of the class. As many of the activities associated with each approach required incubation times beyond the time allocated to the class, groups would often address more than one hypothesis during a given lab meeting. Students used flowcharts of daily activities, in the context of each hypothesis being tested, to stay on track and avoid confusion while multitasking. During these meetings, the instructor served to facilitate the organization and timing of the experiments and lead class discussions of results. The four approaches that students inevitably design are described in the following sections.

Null Hypothesis 1: The Topoisomerase in the TOPO-TA Kit Demonstrates No Observable Preference for the Orientation of Actin cDNA Insert in the Ligation Reaction

The amplified cDNA that is used for the ligation reaction possesses 3′-A overhangs that are added by Taq DNA polymerase. These modified ends are used by topoisomerase to ligate the amplified product to the 3′-T overhangs on a linearized version of the pCR2.1 vector (see review in the Invitrogen TOPO-TA kit user manual [7]). To determine if there is a preference for a particular insert orientation in this ligation reaction, student brainstorming sessions typically lead the use of a PCR-based strategy to identify the presence or absence of each orientation in the ligation products. Further discussion generally leads to the drawback of not being able to accurately quantify the amount of each template using standard PCR since the reactions generally proceed to completion regardless of the differences template concentration. To circumvent this problem, quantitative real-time PCR is introduced and one method, using TaqMan chemistry, is described in detail (for more information about the methods of real-time PCR and a published application, see [9, 10]). Because of the cost of reagents and limited educational value of setting up the reaction, we chose not to have the students perform this experiment and instead discuss how the reaction would be set up (primer and probe design, cycling parameters, etc.) and the proper controls that would be necessary for obtaining conclusive data. Following this discussion, the raw data obtained from an experiment performed outside of class is presented. Data from three separate ligation reactions were assayed to determine the relative abundance of pACT-S and pACT-A plasmids among the ligated products (see method section in the online supplement). It was found that a slight difference was apparent in the ligation reaction (less than one cycle, or a twofold difference in template quantity, see supplementary figure), but students generally agree that the inequality was not sufficient to account for the complete absence of pACT-S in the accumulated student samples. The data from this relative qPCR assay supported the null hypothesis, in that students could not attribute the observed bias to a selection for or against one of the two possible recombinant plasmids at the level of the topoisomerase ligation reaction.

Null Hypothesis 2: There Is No Cellular Toxicity Associated with the Sense Orientation of the Actin cDNA

Before addressing how toxicity may be involved, student must first recall information that was previously covered in the class. First, they should reexamine the genotype of the recipient strain for the transformation (TOP10, see genotype description on Invitrogen site [7]). This strain possesses the lacX74 mutation, which completely removes the endogenous lac operon, including the lac repressor (lacI). Without this repressor, the regulatory region controlling the expression of the lacZ′ in pCR2.1 would no longer be regulated by the inducer (e.g. allolactose or IPTG). Second, during an earlier computer lab exercise, it was also determined that the actin cDNA coding region is in frame with the lacZ′, with no stop codon that would interrupt the expression of this gene in the TOP10 strain. A brief search of the literature supports the idea of actin toxicity, as it has been found that the expression of eukaryotic actin in bacteria can result in the formation of protein aggregates [11]. As the unhindered expression of a fusion protein between the amino terminal portion of the lacZ alpha-fragment and yeast actin may form similar aggregates, the possibility of toxicity associated with this orientation merited further investigation.

To determine the role of toxicity on the growth of cells carrying pACT-S, students proposed to change the host strain to one that expressed the lacIq allele (TOP10-F′, see genotype in [7]), which would turn off the expression of the lacZ′ promoter and thus the expression of the cloned actin insert. The effect of toxicity could then be evaluated by exposing the cells to an inducer (e.g. IPTG), which would activate the expression of lacZ′-actin fusion protein. The TOP10-F′ cells were similarly transformed with the TOPO-TA ligation products generated earlier in the class and plated onto selective medium in the absence of the inducer. Colonies arising from this transformation were then replica printed onto plates with or without IPTG to identify transformants that only formed colonies in the absence of IPTG, indicating that the fusion protein expressed under IPTG induction inhibited growth. For a more detailed description of replica printing and a published application of this technique, see [12, 13]. Students found that there was no apparent difference in growth between the plates, suggesting that the impact of toxicity was not detectable in this assay. However, it was observed that the lacIq inhibition was incomplete as several light blue colonies were observed on plates containing the X-gal substrate but no IPTG inducer. Although no support for the presence of toxicity was identified, one could argue that this incomplete inhibition of the lacZ′ expression may have prevented the formation of colonies prior to the replica plating survival assay. As such, there was some discussion between the students as to whether they should refute the null hypothesis or state that no conclusion could be made from these results. This proved to be an invaluable learning moment for the students in the importance of controls (such as the replica print onto plates without IPTG, yet containing X-gal) and how they may be used to evaluate the reliability of results.

Null Hypothesis 3: The Insert Orientation Has No Effect on the Replication Competence of pCR2.1

As in hypothesis 2, some background information is required to help students identify how the insert orientation might affect plasmid replication. Plasmids are traditionally designed so that all of the inserted gene cassettes (including the selectable antibiotic resistance genes) are arranged in a head-to-tail format. This arrangement is also observed frequently in naturally occurring plasmids and even in the genomic organization of many prokaryotic cells [14]. It has been observed that in plasmids bearing unidirectional origins, the organization of gene cassettes affects plasmid replication and thus the inheritance of those plasmids in dividing cells. The ColE1 origin of replication used in pCR2.1 is one of the more commonly used unidirectional origins and has been shown to be adversely affected by bacterial transcriptional units that proceed counter to the direction of the replication complex, resulting in plasmid instability [15, 16]. Although our yeast cDNA insert would not be expected to contain a bacterial promoter, on further investigation in the computer lab, elements closely resembling the -10 and -35 sigma factor binding sites were identified on the anti-sense strand of ACT1 (see Fig. 3a). This observation was particularly interesting in that the promoter element would only be directed counter to the ColE1 origin in those plasmids bearing the sense orientation (pACT-S). Students proposed that this promoter could reduce the replication potential of the plasmid to such a degree that growth is inhibited and detectable colonies would not form. To test this hypothesis, students proposed to remove the promoter elements from actin using site-directed PCR mutagenesis, a topic that was covered earlier in the class when describing alternative uses of PCR (for information on site-directed PCR and a published application, see [17, 18]).

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Figure 3. Identification and site-directed PCR mutagenesis of putative sigma factor binding sites identified in yeast ACT1. (a) Comparison of the sequences corresponding to the consensus -10 and -35 elements in prokaryotic promoters, the putative elements found in yeast ACT1, and the silently mutated sequence following the generation of the SigMut version of ACT1. (b) Construction of the SigMut allele is illustrated in the agarose gel assay of the products from each step. Lanes in this gel represent: (1) the 1Kb DNA ladder (Promega); (2) PCR product of ACT1 5′ region (mutated -10 element); (3) PCR product of ACT1 3′ region (mutated -35 element); amplification of 5′ region (4), 3′ region (5), and combined 5′ and 3′ products (6) using ACT-F and ACT-R primers that were used to amplify complete ACT1 cDNA. The absence of products in lanes 4 and 5 indicate that the product of lane 6 is dependent on both fragments being included in the reaction.

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To separate the possible effect of toxicity (hypothesis 2) from plasmid replication competence, primers were designed that would specifically mutate the two promoter elements while maintaining the translated amino acid sequence of yeast actin (see Fig. 3a). The mutagenic primers (SigMut-F and -R), combined with the original primers used to amplify the cDNA, were used to separately amplify two regions of the cDNA that overlap by 14 nucleotides. The two PCR products were gel purified, denatured, and combined to amplify a complete ACT1 cDNA bearing the mutations using the original two actin primers (see Fig. 3b). The presence of an additional AluI site associated with the site-specific mutations (referred to as the SigMut allele) was used to confirm the presence of the introduced mutations in the amplified product. The amplified SigMut allele of ACT1 was ligated into pCR2.1 using TOPO-TA, as done previously. To quickly and easily screen through multiple recombinant colonies for the presence of pACT-S, colony PCR followed by a diagnostic digest with HindIII was performed. As with the wild-type actin cDNA, all of the white colonies screened were found to still possess only the pACT-A version of the plasmid. Students concluded that these data do not refute the null hypothesis: that plasmid replication interference does not appear to be the basis of their inability to recover pACT-S among the selected recombinant clones.

Null Hypothesis 4: The Alpha-Complementation Screen for Recombinant Plasmids Effectively Discriminates Between Plasmids Derived from Intramolecular and Intermolecular Ligation Events, Regardless of the Insert Orientation

Beta-galactosidase is a common reporter used in molecular biology to distinguish between self-ligated plasmids (intramolecular ligations) from recombinant plasmids (intermolecular ligations that incorporate exogenous DNA). This strategy arose by the observation that the coding region for the beta-galactosidase gene can be separated into two discrete transcriptional units that produce separate polypeptide fragments of the enzyme (alpha- and omega-fragments), which interact in the cytoplasm to produce a functional enzyme [19]. The alpha-fragment is traditionally expressed from a plasmid (lacZ′) whereas the omega-fragment gene resides within the host cell genome (lacZΔM15). By engineering an MCS within the coding region of lacZ′, it is assumed that any interruptions from the ligation of a DNA within the MCS would disrupt the alpha-fragment and thus the beta-galactosidase activity. The enzyme activity is monitored through the use of a synthetic substrate (X-gal) that is added to the growth media. If beta-galactosidase activity is expressed, X-gal is cleaved and forms an insoluble indigo precipitate that stains the colony blue. If the alpha-fragment is no longer functional or able to associate with the omega-fragment, the enzyme activity is abolished and the colony is colorless (white).

Although it is expected that insertion of the 1.2-Kbp yeast actin cDNA would disrupt this activity, regardless of its orientation, students proposed the possibility that pACT-S maintains the expression of a functional alpha-fragment. As such, the assumption that blue colonies only possess plasmids arising from intramolecular ligation events would result in pACT-S being left behind. On a closer examination of the results from their original transformation plates, students recalled that three discrete morphologies were observed: white, light blue, and dark blue at a consistent 10:10:1 ratio, respectively. To examine the nature of the plasmids that were propagated in each of the classes, all three were targeted for a colony PCR and digest screen as described in hypothesis 3. In a representative set of data screening through 56 colonies from a single transformation, the white colonies were found to only possess the pACT-A, the dark blue colonies were empty vectors, and surprisingly, many of the light blue colonies carried the elusive pACT-S (see Fig. 4). These results support the alternative hypothesis, which states that the alpha-complementation screen for recombinant plasmids does not properly discriminate between plasmids derived from intramolecular and intermolecular ligation events and that selection of only white colonies is likely to have contributed to the bias against clones containing pACT-S. A further investigation of the effect of toxicity and plasmid replication on this bias is described in the online supplement associated with this article.

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Figure 4. Infidelity identified in alpha-complementation screen using colony PCR and restriction mapping. (a) Example gel electrophoreses of HindIII digestion products of PCR-amplified DNA from a white (lane 2), light blue (lane 3), and dark blue (lane 4) colonies using primers that flank the MCS of pCR2.1. White colonies produced fragments that corresponded to the bands expected for pACT-A (1 Kbp and 425 bp) while those from light blue colonies produced bands representing pACT-S (880 bp and 614 bp). The less frequent dark blue colonies, containing plasmids that did not contain an insert (empty pCR2.1), generated a band at 300 bp. The 1-Kb DNA ladder (Promega) is shown in lane 1. (b) Data obtained from screening colonies from an LB-AX plate of TOP10 transformed with the TOPO-TA ligation products of amplified wild-type ACT1 cDNA.

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Student Assessment

Student learning outcomes (e.g. proficiency in the use of molecular techniques and the development of investigative critical thinking skills) were assessed by both formal and informal methods. Midterm and final examinations were used to test student understanding of the techniques and how they may be used to solve novel problems that were posed on the exams. A take-home exam format was used for the comprehensive final exam to allow students to brainstorm experimental approaches and use programs introduced in the computer lab to address problems. Weekly, unannounced lab quizzes were given in class to assess student preparation for the activities of the day and how these activities relate to the overall experimental design that is being followed. At the start of new experiments, students were also required to prepare illustrated flowcharts that accurately represented each step of the written procedures found in their lab manual. Throughout the quarter, our students were required to maintain a detailed lab notebook that included experimental goals and expectations, procedures, list of reagents and equipment used, results, and a conclusion/discussion section. The importance of maintaining a proper notebook was reinforced throughout the investigative portion of the class as students were required to reevaluate their previous notebook entries to complete the project. Rubrics were fashioned for the notebooks, and unannounced inspections were performed throughout the quarter to encourage continued maintenance. Informal self-assessments of their abilities and opinions of the class were evaluated through anonymous surveys provided in our course Blackboard page. Students were asked to evaluate their ability to perform the following list of skills during the first and last week of class:

  • S1: Isolate one or more of the various types nucleic acids (e.g. RNA, gDNA, plasmids)

  • S2: Evaluate nucleic acids by agarose gel electrophoresis

  • S3: Use the polymerase chain reaction to amplify DNA

  • S4: Design the sequence of a set of primers to amplify a particular target region

  • S5: Use computer programs to analyze DNA sequences

  • S6: Address a novel problem by designing and executing a set of experiments involving molecular techniques.

Students were asked to rank themselves using a scale from 1to 4, with 1 = no experience, 2 = limited experience, 3 = completed more than once, and 4 = able to complete the task independently. Significant improvements in student self-perception were obtained in all six skills, with most expressing confidence in their ability to complete all of them independently by the end of the 10-week course (see Fig. 5). All students surveyed in two lab sections (33 responses recorded) stated that the investigative approach was challenging but should be maintained in future course offerings. In an open-ended question concerning the most valuable aspect of the class, 24% stated that this investigative approach was the most useful portion of the class, 60% indicated that the greatest value was learning the particular techniques, and the remaining 16% were undefined, stating that the entire class was of great benefit. When asked to express their views concerning the value of the investigative approach used for the last 3 weeks of class, the following responses were typical of those who completed the survey:

  • “I really enjoyed the opportunity to think of the hypothesis ourselves and really investigate them, rather than just follow what the book says like most labs.”

  • “The techniques learned in the first part of the class are an invaluable practical learning experience, applicable for any sort of mo bio lab job. The project approach section of the class reinforces those skills learned in the first half of the class and challenges the student to think about the concepts more rigorously and critically.”

  • “It forced me to take a good notebook, use information that I learned in other classes, use information I learned in this class in new ways, and it was a really good look into how research actually works.”

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Figure 5. Skill survey. Self-assessment results from students surveyed at the start (grey bars) and end (black bars) of our molecular biology laboratory course. Students were asked to rank themselves on the ability to complete six different skills (see text) using a scale from 1–4, from no experience (1) to being able to complete the task independently (4). Data were accumulated from two classes, with a total of 33 students responding (94% of the enrolled students). Error bars represent the standard deviation from the mean.

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Although some of the students mentioned the need for more structure in the investigative section of the class, this is a common student response when problem-based approaches are used in place of a more traditional laboratory class [20]. It was apparent, however, that teamwork was essential as confusion arose in groups when there was a breakdown in communication or loss of focus during meetings when multitasking and the delegation of responsibilities were required.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The primary objective for any molecular biology laboratory course should be to prepare students for the research environment. This course design is distinct among many of the labs that students take at Cal Poly, as each meeting progresses toward a central goal of the class: to construct and isolate the pACT-S plasmid. By specifying this particular orientation, the class is set up to encounter a problem after screening through the selected recombinant clones. The failure to obtain the pACT-S plasmid invokes an opportunity to have students deliberate the possibilities, pose hypotheses, and design experiments to address the cause of the problem. This type of problem-based approach for laboratory activities serves a critical need in the sciences: the ability to critically think and troubleshoot failed experiments. Our class meets the three teaching formats described by Boyer: skill-building, project-based, and inquiry-based approaches [21]. The first section focuses on skill building but in a project-based format with preset schedule that does not waiver from a plan introduced to students at the beginning of the class. The second section is a modification of this plan because of a problem that arises as students find they are unable to accomplish the goal. As a result, students must design plans to discover why this was not possible. This section of the class is in actuality a combination of an inquiry and project-based approach as the students will inevitably (with some guidance, if necessary) arrive at the same four hypotheses and experimental designs to address them. As with most of the research projects, there is generally a limited number of hypotheses that are investigated to approach a given problem. By subtly encouraging the students to use what we have learned in class to address each hypothesis, this modification of an independent project cuts down on reagents/resources that the instructor requires without sacrificing the process of student-led inquiry.

Our students have commented on their excitement in designing projects and using their newly attained lab techniques to solve a “real world” problem. They have also been enthused by their results and conclusions, which they discuss in class during the last meeting. Often, students are trained in laboratory techniques on projects that either consistently provide them with the expected results or fail without the opportunity to determine the cause of the malfunction. As opposed to concluding that the plasmid bias was due to student or technical errors (a common cause of failed experiments), students were satisfied with the more interesting conclusion that the inappropriate expression of beta-galactosidase in pACT-S strains was primarily responsible for the failure to initially accomplish the goal. Through this series of experiments, students learn about the effect of gene expression, plasmid replication, and problems that may arise when assuming traditionally accepted screens, such as the alpha-complementation, always work as expected. Through repetition and novel application of foundational molecular lab techniques, they leave the class more confident in their ability to complete these techniques on their own and even design a series of experiments to solve a new problem. In addition, we believe our students learn a valuable lesson: to move beyond their background knowledge and traditional wisdom and to place more trust in their experimental design and interpretation.

ADDITIONAL PROJECT QUESTIONS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
  • 1)
    Is there a difference in the beta-galactosidase activity between the sense orientation clones obtained from wild-type actin and those with the SigMut mutations? Note: this would be best completed as part of a growth curve assay in liquid cultures with the ONPG substrate.
  • 2)
    Is the blue color of the sense orientation dependent on the downstream carboxyl-terminus region of the lacZ′ gene, despite the presence of the actin stop codon? Note: this may be determined by performing a PstI digest of the pACT-S plasmids with a self-ligation to remove this region (preliminary data suggest that removal of this region eliminates the expression of beta-galactosidase activity).
  • 3)
    Would the same level of bias be observed if the intron-bearing gDNA PCR product were cloned in place of the cDNA? Note: the intron sequence begins following the 11th nucleotide in the actin coding sequence and contains multiple stop codons in the lacZreading frame.
  • 4)
    Is there a difference in the number of plasmids maintained in cells bearing the cDNA insert in the sense as compared with the anti-sense orientation? If there is a difference, is it eliminated on mutating the sigma binding site described in the third hypothesis? Note: this would include a nice experiment to introduce students to blotting techniques that quantify the plasmid load maintained in cell preparations of known cell density.

Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We acknowledge the technical support of John Merriam and Alice Hamrick in our Biological Sciences Department and Lisa Munding in the Undergraduate Biotechnology Laboratory facility at Cal Poly.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. CONCLUSIONS
  6. ADDITIONAL PROJECT QUESTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1470-8175/suppmat .

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