Gene Amplification by PCR and Subcloning into a GFP-FusionPlasmid Expression Vector as a Molecular BiologyLaboratory Course*
Received for publication, October 27, 2003, and in revised form, January 9, 2004
Joshua A. Bornhorst‡§¶, Michael A. Deibel�, and Amy B. Mulnix‡**
From the ‡Department of Biology, Earlham College, Richmond, Indiana 47374; §Department of Pathology,University of Utah, Salt Lake City, Utah 84108, ¶Associated Regional and University Pathologists, Salt Lake City,Utah 84108; and �Department of Chemistry, Earlham College, Richmond, Indiana 47374
A novel experimental sequence for the advanced undergraduate laboratory course has been developed atEarlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment,undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in abacterial expression plasmid over the course of a single semester in a weekly 3-h laboratory period.Students designed PCR primers for amplification of the selected gene using computational DNA sequenceanalysis tools. During the experimental portion of the course, students amplified and ligated the target DNAinto a commercially available GFP expression vector. Following transformation of the ligation product,plasmids were harvested from the resulting bacterial colonies and were analyzed by restriction digestionto confirm the creation of the chimeric GFP-DNA. This course gave students valuable experience withcommonly used molecular techniques in an authentic research project. In addition, students gainedexperience with experimental design and execution. The techniques presented here are flexible and can begeneralized for use with almost any DNA sequence and expression vector. This series also serves as anexample of how faculty can adapt their ongoing research projects to the undergraduate laboratory.
Keywords: PCR, subcloning, green fluorescent protein (GFP), affinity tag, serpin.
Generating recombinant DNA that, when expressed,yields a fusion protein is a common approach for investi-gating protein function [1]. The addition of a domain to aprotein sequence can serve as a molecular reporter, allow-ing detection of a protein in subsequent experiments. Forexample, the intrinsic fluorescence of green fluorescentprotein (GFP)1 upon excitation with ultraviolet light is usedto visualize the cellular location of a target protein to whichGFP has been fused [2]. The addition of a small epitope tagis also useful to study protein function because it facilitatespurification by affinity chromatography. One such epitopetag consists of six consecutive histidine residues attachedat either the amino or carboxyl terminus of the targetprotein. This molecular handle allows for rapid and efficientisolation of the tagged protein from an expression hostbased on the histidines’ affinity for a nickel column [3].
Motivated in part by the national reform movement toinclude discovery-based learning in courses and laborato-ries [4–6], we created an experimental series for a semes-ter-long laboratory course that engineered an epitope-
tagged, chimeric protein containing a GFP marker.Sciences at Earlham College, a private undergraduate in-stitution, have a long-standing tradition of incorporatingdiscovery-based projects into laboratories beginning in thefreshman year. Students become progressively more in-dependent in biology and chemistry laboratory workthroughout their college careers. The 2003 course in whichthis series was used, Advanced Cell Physiology Labora-tory, enrolled students ranging from sophomores to se-niors. Enrollment in the course was unusually small, withonly four students.
Project Kaleidoscope (PKAL) asserts [4] that a strongundergraduate program is created when “learning is ex-periential and steeped in investigation, . . . [when] learningtakes place in a community where faculty are committedequally to undergraduate teaching and to their own intel-lectual vitality, where faculty see students as partners inlearning, where students collaborate with one another andgain confidence . . . and where institutions support suchcommunities of learners.” Well-designed summer researchexperiences for undergraduates can create environmentssuch as those described by PKAL. Building such researchexperiences into regular coursework is more challenging.
We envisioned the generation of a fusion protein as aseries of experiments easily adapted to an undergraduatelaboratory course. Such a series creates abundant oppor-tunities to engage students in an authentic research expe-
* This work was supported by Howard Hughes Medical Insti-tute Grant 52002642 to Earlham College.
** To whom correspondence should be addressed: Departmentof Biology, Earlham College, Richmond, Indiana 47374. Tel.: 765-983-1498; Fax: 765-983-1497; E-mail: [email protected].
1 The abbreviations used are: GFP, green fluorescence protein;PKAL, Project Kaleidoscope.
This paper is available on line at http://www.bambed.org 173
rience; one including experimental design, data interpre-tation, and trouble-shooting protocols, as well as one thatlinks a series of molecular techniques in the pursuit of asingle experimental goal. Because the project had an un-certain rather than a predetermined outcome, we also sawthe generation of a fusion protein as a collaborative effortamong faculty and students. The project was designed notjust to model as closely as possible a research experience,but also to advance the research projects of Dr. Mulnix.
In the Advanced Cell Physiology Laboratory course, stu-dents constructed a His-tagged, GFP-fusion serpin. Ser-pins [7] are a family of small (Mr 45–70 kDa) proteinsinvolved in a variety of vertebrate homeostatic processesincluding blood clotting, wound repair, extracellular matrixremodeling, inflammation, and the immune response. Theyregulate these processes by inhibiting the activity of theserine proteases involved in the reaction cascades. Kanostet al. identified the first insect serpin in Manduca sextabased on an amino acid sequence deduced from thecDNA [8]. They also demonstrated inhibitory activityagainst elastase of the purified cognate hemolymph pro-tein. In vertebrate systems, the stable serpin-enzyme com-plex is removed from circulation via receptor-mediatedendocytosis. The fate of serpin-enzyme complexes in in-sects remains unknown. The GFP-serpin fusion is a steptoward investigating the possible clearance of serpin-en-zyme complexes from insect hemolymph. The project goalwas to construct a gene encoding a GFP-His6-taggedserpin using commercially available vectors and plasmidscontaining M. sexta serpin cDNA clones to which His tagshad previously been added [9]. In our case, the GFP proteinwas added to the N terminus of the His6-serpin because ashort section of the C terminus is cleaved and released uponformation of the stable serpin-enzyme complex.
Although this article focuses on the laboratory course,the experimental series presented here also was used by afaculty-student collaborative project. Under the directionof Dr. Deibel, a group of four students generated a recom-binant transferrin gene during a semester-long independ-ent study project. Members of the transferrin family, whichare single-chain glycoproteins of �80 kDa that tightly bindFe under physiological conditions, are found mainly in theserum where they transport iron [10, 11]. The goal of thisproject was to add a His6 tag and an enterokinase prote-
ase recognition site to the N-terminal lobe of transferrin.The enterokinase recognition site enables the cleavage ofthe desired protein product from the His6 tag followingpurification. Creation of the gene fusion facilitates produc-tion of large amounts of protein for functional assays andsubsequent incorporation of labels into the protein (e.g.fluoridated amino acids) to help study the structural effectsof oxidative damage. Use of the presented experimentalseries in a second context demonstrates that it can beadapted to a wide variety of biological inquires in either anindependent study or summer research setting.
The general experimental series presented here is par-ticularly well suited for use in undergraduate education. Itintegrates commonly used molecular techniques such asplasmid DNA isolation, PCR, restriction enzyme digestion,gel electrophoresis, and transformation, all of which ourstudents had been exposed to from their introductorycourses. Because recent advances in commercially avail-able kits have dramatically reduced the time needed toperform these techniques, experimental manipulations canbe designed to fit into a typical 3-h laboratory block andthe materials can be stored safely until the following week.Furthermore, the availability of vendors to quickly synthe-size primers and perform DNA sequence analysis allowedrapid and relatively inexpensive turn around at criticalpoints. Thus, the experimental framework described hereallows students and faculty to complete a research projectof their unique design in either a semester-long independ-ent study format or a laboratory course (see Table I).
EXPERIMENTAL PROCEDURES
General Cloning Strategy—A widely used method to generaterecombinant genes involves amplifying the gene of interest byPCR and then ligating the PCR product into the desired plasmidvector. In order to facilitate this process, PCR primers can bedesigned to incorporate appropriate restriction sites at the endsof the target DNA for subsequent digestion and ligation into thevector of choice. The addition of restriction sites by PCR duringamplification of a genetic sequence allows for the insertion ofvirtually any target gene into any vector [12].
This cloning strategy was used to create a recombinant M.sexta GFP-His6-serpin cDNA construct inserted into the expres-sion vector pGFPuv (Clontech, Palo Alto, CA) (Fig. 1A). Specifi-cally, a M. sexta-mutagenized cDNA clone of functionalserpin1B(A343K) previously engineered with a His6 tag at the Nterminus of the protein was used as a template in PCR synthesis
TABLE ISuggested weekly class schedule in a 3-h laboratory course
1 Transformation of serpin cDNA into DH5�2 Plasmid DNA isolation and student-designed diagnostic restriction
digestSet up overnight culture on the day prior to lab
3 E-gel of cut vector and primer design4 Primer design and primer ordering Reconstitute PCR primers5 PCR of gene of interest6 Use of E-gels to check PCR product restriction digestions Store PCR product at –20 °C following PCR7 Restriction digestion of plasmid and vector. CIAP treatment of vector.8 Ligation and transformation of ligation product9 Plasmid preparation DNA from transformants; diagnostic digestion of
transformant plasmid DNASet up overnight cultures of transformants
10 Agarose gel of diagnostic DNA digestion to identify recombinantproduct; design and order sequencing primers
11 DNA sequencing Reconstitute sequencing primers12 Interpretation of sequencing results Open sequence data files
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(Fig. 1B) [9]. The PCR primers were designed to add a 3� (codingstrand) EcoR1 site and a 5� (coding strand) SacI site to theHis6-serpin sequence (Fig. 2A). Both the His6-serpin PCR productand the pGFPuv expression vector were digested by SacI and
EcoRI. The His6-serpin PCR product then was inserted at the 3�end of the GFP coding sequence using the 5� multi-cloning site ofthe vector (Fig. 1C). The final chimeric protein carries GFP at theN-terminal end, a His6 affinity tag flanked by flexible glycine
FIG. 1. Overall cloning scheme to produce a GFP-His6-serpin construct. A, a map of the plasmid pGFPuv, which was purchasedfrom Clontech. All plasmid maps were created by the shareware program MacPlasMap (v1.83). The GFPuv and ampicillin resistancegenes are represented as black arrows. The arrows point in the direction of protein translation. The inducible lac promoter Plac controlsGFPuv expression. The pUC origin of replication maintains a high copy number of the plasmid in the cell, facilitating plasmidpurification. The multi-cloning sites (MCS) are shown in gray. The locations of the translational start and stop sites of the GFPuv gene,as well as the unique restriction sites SacI and EcoRI, are shown. The pGFPuv plasmid is digested with SacI and EcoRI and treatedwith the phosphatase CIAP, yielding a linearized DNA fragment that cannot relegate to form a circular plasmid. The sticky ends createdby the restriction digestion of pGFPuv are used to ligate complementary sticky ends of the PCR insert containing the serpin cDNA. B,the PCR amplification product generated from plasmid serpin1B(A343K) containing the His6-serpin cDNA. The PCR primers aredesigned to introduce the complementary restriction sites to the ends of the His6-serpin DNA. These restriction sites must be uniquein the PCR amplification product. Digestion of the PCR amplification product with SacI and EcoRI generates sticky ends suitable forligation into the similarly linearized pGFPuv vector. C, the GFP-His6-serpin expression vector pMAJILK created by ligation of thelinearized pGFPuv and the digested PCR amplification product. The complementary sticky ends created by the two restrictionenzymes ensure proper directional ligation of the insert into the vector. The translation of the resulting 1.9-kb GFP-His6-serpin geneis under control of the inducible lac promoter. D, the protein product created by translation of the GFP-His6-serpin gene. This 75-kDaprotein consists of a N-terminal 27-kDa GFPuv domain followed by a flexible linker domain that includes the His6 sequence. TheC-terminal serpin domain is 45 kDa and contains the C-terminal protease cleavage site. The C-terminal protease cleavage sitemandated that the GFP domain be added N-terminal of the serpin domain. The structures of the isolated GFP and serpin domains arealso given. As shown, the figure is only approximately to scale. The flexible linker between the GFP and serpin domains consisting ofglycine residues on either side of the His6 domain may promote accessibility of the epitope tag to solvent. Although the exact locationof the domains in the recombinant protein is not known, the flexible linker appears to allow these domains to adopt positions that donot functionally interfere. GFP (#1EMA) and M. sexta serpin (#1SEK) files were downloaded from the Protein Data Bank andmanipulated using Deep View-Swiss Pdb Viewer (us.expasy.org/spdbv). The protein is depicted in standard ribbon diagram format.The linker region between the two proteins is shown as a linear B-strand to demonstrate the potential three-dimensional relationshipof the individual domains.
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FIG. 2. Primer design and PCR of the His6-serpin cDNA. A, overview of primer design for PCR amplification of the His6-serpingene. Shown is the double-stranded 1.2-kb His6 serpin cDNA in plasmid serpin1B(A343K). The large arrow denotes the direction oftranscription. Following heat separation of the two strands, two synthetic oligonucleotide primers are added to serve as templates forPCR amplification. These primers consist of a 3� complementary and 5� noncomplementary region. The 3� complementary regions ofthe primers serve as the initiation points for primer elongation, while the 5� noncomplementary regions allow for introduction ofrestrictions sites to the PCR product. The front primer binds to the noncoding strand near at the start of the His6-serpin gene, and theback primer binds to the coding strand at the end of the His6-serpin gene. B, design of the front DNA primer. Shown is the front primerbound to the noncoding strand of the His6-serpin gene in the plasmid serpin1B(A343K). Single-letter amino acid abbreviations for each
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residues in the middle of the construct, and the serpin cDNA atthe C-terminal end (Fig. 1D).
Sequence Management—A computer program was usedthroughout the course to document and manipulate DNA se-quences, including generating restriction maps and translatingDNA sequences into protein sequences. While many sequencemanagement programs are available, the shareware programDNA Strider 1.1 (Macintosh) was utilized [13]. In many cases,complete plasmid sequences can be downloaded from commer-cial websites and manipulated by these programs. The use ofsuch a DNA sequence management program, while not required,does familiarize students with DNA manipulation and databasemanagement. Sequence management programs are extremelyhelpful in developing initial cloning strategies.
Organization of the Laboratory Series—Our students workedindividually, with each one performing their own set of reactionsthroughout the semester. They did however engage in a greatdeal of collaboration and support, sharing materials, discussingexperimental options, coordinating agarose gel use, etc. A facultymember was present throughout the laboratory period, providingguidance and appropriate intervention. The laboratory period wasa 3-h block one afternoon a week. On several occasions, stu-dents were asked to perform brief preparations (e.g. inoculatecultures) on the afternoon before our laboratory session. A pro-posed timeline of laboratory exercises over the course of thesemester is shown in Table I.
Week One
Transformation—An M. sexta serpin cDNA serpin1B(A343K)was previously cloned into the expression plasmid pQME-60,which added DNA encoding a His6 epitope tag to the N-terminalend of the protein to create the plasmid serpin1B(A343K) [9]. Inprinciple, any plasmid, genomic DNA isolation, or cDNA librarycould serve as the template for PCR at the outset of this exper-iment. Our students performed separate transformations of theplasmid serpin1B(A343K) and pGFPuv into Escherichia coli-com-petent cells. During all genetic manipulations, a stable E. colicloning strain lacking the recA recombination gene, such as XL-1Blue (Stratagene, La Jolla, CA) or DH5� (Invitrogen, San Diego,CA), was used. Subcloning-grade competent cells yielding �1 �106 transformants per microgram of DNA were obtained fromcommercial sources. The transformation protocol recommendedby the competent cell manufacturer took under 2 h, and theresulting cells were plated on ampicillin-containing plates. Theseplates were kept at 37 °C overnight (12–16 h) to allow sufficientbacterial colony growth. On the day following the transformation,students returned to the laboratory removed their plates from theincubator, noted their results, and stored their plates at 4 °C forthe intervening period. The students were instructed to calculatethe number of colonies per microgram of DNA for their reactions.
A negative control (no plasmid DNA) and a positive control
(plasmid DNA from a commercial source, e.g. pUC18) were in-cluded for the transformation. Our students used pre-preparedwarm autoclaved Luria-Bertani (LB)/agar solution and pouredplates at the beginning of the laboratory period. A suitableamount of 1000� stock of ampicillin solution (0.1g ampicillin/mlof 50% ethanol/water solution, store at �20 °C) was added to theliquid agar prior to pouring.
Week Two
Plasmid Preparation—On the afternoon prior to the secondlaboratory period, students started small (2 ml) cultures of LBampicillin broth from a single colony on their transformationplates. The culture was incubated at 37 °C with shaking (225 rpm)overnight. Students performed this inoculation in �15 min; alter-natively, the faculty or teaching assistant can begin these cul-tures. Incubation of the cultures should not exceed 24 h to ensurethe plasmids are produced in healthy cells.
During the laboratory session, plasmid DNA suitable for PCRwas generated from this culture using a miniprep DNA isolation kit(available from Qiagen at a cost of about $1 per plasmid prepa-ration). The miniprep procedure should take the average student�1 h and yield 50 �l of purified plasmid at a concentration of�200 ng/�l.
Restriction Digests—In order to confirm that the plasmid prep-aration was successful, a diagnostic restriction digest was per-formed. Students were given a plasmid map and asked to designa restriction digestion that gave a fragment pattern characteristicof the correct plasmid. For example, digesting the pGFPuv vectorwith the enzyme SacI or EcoRI should yield a single linearizedDNA fragment of 3.3 kb as visualized on an agarose gel. Theseenzymes and digestion protocols were obtained from commercialsources (e.g. New England Biolabs, Promega). Generally, 200 ngof purified plasmid DNA were digested in a total volume of 10 �l.The digestion can readily be scaled up to digest larger amounts ofDNA. The volume of enzyme needed was calculated using theactivity of each enzyme preparation as indicated by the manu-facturer. An incubation of 30 min is usually sufficient for anappropriate amount of enzyme to specifically cut the majority ofthe DNA. If desired, more complex digests can be performedutilizing more than one enzyme simultaneously. If this option ispursued, students must select a digestion buffer and reactionconditions that are compatible with all of the enzymes. The di-gested DNA fragments were stored at �20 °C until the followingweek.
Week Three
DNA Fragment Visualization by Electrophoresis through anAgarose Gel—Agarose E-gels (Invitrogen) provide a safe andrapid method to visualize the DNA fragments. The gels do notrequire electrophoresis buffer and run in less than 45 min. The
codon are shown above the coding strand. The 5� noncomplementary end of the primer consists of the SacI restriction site and a6-base overhang of random sequence to allow efficient cleavage of the SacI site. As the SacI site in the plasmid pGFPuv lies just beforethe end of the GFPuv gene, DNA sequence coding for the last two amino acids in GFPuv was added subsequent to the restriction siteto maintain the full-length GFPuv gene upon subcloning of the PCR product into pGFPuv. The bases of the noncomplementary regionof the plasmid are denoted as N. A glycine residue was also introduced to allow conformational flexibility of the pGFPuv domain relativeto the rest of the gene product. The cleavage site of the coding DNA strand upon digestion with SacI is shown. The complementaryregion of the primer consists of the start of the His6-serpin gene and included an alanine linker residue prior to the start of the serpindomain. Periods represent long DNA sequences extending from the sequence shown. The arrow indicates the direction of primerextension in a PCR. C, design of the back DNA primer. Shown is the back PCR primer bound to the coding strand of the terminus ofthe His6-serpin gene in the plasmid serpin1B(A343K). Single-letter amino acid abbreviations for each codon are shown above thecoding strand. The 5� noncomplementary end of the primer consists of the EcoRI restriction site and a 6-base overhang of randomsequence to allow efficient cleavage of the EcoRI site. The bases of the noncomplementary region of the plasmid are denoted as N.A glycine residue was also introduced to allow conformational flexibility of the pGFPuv domain relative to the rest of the gene product.The cleavage site of the coding DNA strand upon digestion with EcoRI is also shown. An additional stop codon was introduced afterthe endogenous stop codon to ensure termination of the gene product. Periods represent long DNA sequences extending from thesequence shown. The arrow indicates the direction of primer extension in a PCR.
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agarose gel containing the fluorophore ethidium bromide issealed between two plastic plates, thus minimizing student ex-posure to this carcinogen. In addition, no DNA loading buffer isrequired, streamlining the loading process. Alternatively, a stand-ard agarose gel that is poured by the students and appropriatelystained to visualize the DNA can be used. Following electrophore-sis, the DNA fragments were visualized using an ultraviolet lightsource. An example of a 1.2% E-gel on a transilluminator isshown in Fig. 3. Approximately 200 ng of DNA were loaded ineach lane (equivalent to about 1 �l of DNA preparation). This
amount of DNA gave a readily apparent band. Larger amounts ofDNA may be needed to see multiple bands or smaller DNAfragments (less than 1.0 kb in length). The linearized DNA wascompared with a DNA ladder (Sigma or New England Biolabs) toascertain that a fragment of the correct length was obtained.Uncut plasmid DNA yields a pattern of multiple DNA bands thatdo not directly correspond to the true length of the plasmid due todifferential DNA supercoiling of the circular plasmid; this was auseful control for determining if the experimental DNAs were fullycut.
Weeks Three and Four
Primer Design—In order to amplify the His6-serpin cDNA se-quence from the plasmid serpin1B(A343K), customized PCRprimers were designed. With the help of the instructors, studentsdesigned and ordered PCR primers that introduced restrictionsites to the ends of the cDNA. The primers consisted of twodistinct regions: the 3� ends of the primers are complementary tothe cDNA and served as the template for polymerase extension.The 5� ends of the primers are noncomplementary to the serpincDNA and allowed introduction of specific DNA sequences to theend of the PCR product (see Fig. 2A).
The “front” primer was designed to introduce a new SacI site inthe PCR serpin cDNA amplification product, which allows forligation to the SacI site in the pGFPuv vector (see Fig. 2B). The 3�end of the primer consisted of a region complementary to the 5�end of the coding strand of the His6-serpin cDNA. The Tm of thisinteraction of the primer with the DNA template should be �60 °Cor greater. A general rule of thumb is that a complementary G-Cpair contributes to the Tm by about 4 °C, while an A-T paircontributes to the Tm by about 2 °C. The 5� noncomplementaryregion of the primer included DNA coding for a glycine residueupstream of the His6 tag to allow for the GFP domain to orientitself independently of the His6 tag and the serpin protein. Theaddition of the glycine also eliminated the GFP’s endogenousstop codon, allowing translation of the full-length GFP-serpinsequence. Finally, the 5� end of the primer was designed toreconstruct the last few codons of the GFP gene, add a SacI site,and a DNA overhang to allow cleavage of the SacI site in theHis6-serpin PCR product. Restriction enzymes often require aDNA overhang to efficiently cleave the restriction site [14]. Moreinformation on appropriate DNA overhangs for individual restric-tion enzymes is available from the New England Biolabs (Beverly,MA; www.neb.com).
The “back” PCR primer, designed to bind to the 5� end of theserpin cDNA noncoding strand sequence, introduces an EcoR1site to the serpin cDNA (see Fig. 2C). The 3� end of this primerconsisted of a sequence complementary to the serpin cDNA. Thiscomplementary section ended at the stop codon of the serpincDNA. The noncomplementary portion of this primer (5� end)added an additional stop codon to ensure termination of transla-tion and contained the introduced EcoRI restriction site. Finally,the 3� end of the “back” primer contained a multibase DNAoverhang beyond the EcoRI restriction site.
Over the past decade, the cost of DNA oligonucleotide primershas dramatically decreased and primers can now readily be or-dered online from companies such as Integrated DNA Technol-ogies or Life Technologies and arrive in less than 3 days at a costof approximately $0.50/DNA base for a 100-nmol preparation.Therefore, primers were designed and ordered online in onelaboratory period. They arrived prior to the next weekly meeting.The lyophilized primers were resuspended by vortexing to a con-centration of 100 �M in molecular biology-grade water and sub-sequently aliquoted and stored at �20 °C.
Week 5
PCR Amplification of the Target Sequence—Amplification ofthe target sequence by PCR was performed utilizing the thermo-philic enzyme Pfu Turbo (Stratagene), which has a higher fidelity
FIG. 3. An 1.2% agarose E-gel (Invitrogen) of DNA samplesgenerated throughout the laboratory course. All samples werediluted to 20 �l and loaded directly onto the E-gel. Followingelectrophoresis at 60 V for 45 min, the gel was placed on aultraviolet transiluminator and the image captured using a CCDcamera. Lane 1 contains the Directload wide-range DNA marker(Sigma, St. Louis, MO). The DNA fragment lengths of the markerare given adjacent to the gel. Lane 2 contains a DNA preparationof uncut pGFPuv showing multiple bands due to plasmid super-coiling. Lane 3 contains the pGFPuv preparation cut singly butEcoRI yielding a DNA fragment of 3.3 kb. Lane 4 contains thepGFPuv plasmid preparation digested by SacI. Lane 5 containsthe double digest of pGFPuv by EcoRI and SacI, yielding a singlevisible fragment of �3.3 kb. Lane 6 contains the �1.2-kb PCRamplification of the His6-serpin product generated from plasmidserpin1B(A343K) and using PCR primers that introduced EcoRIand SacI restrictions site to the ends of the PCR products. Lane7 contains the double digest of the PCR product yielding an�1.2-kb DNA fragment with sticky ends. Lane 8 contains thedilute ligation reaction prior to the addition of ligase containingdigest pGFPuv and PCR product. The PCR product is not visiblein the diluted sample due to its small size. Lane 9 contains thedilute ligation reaction solution following ligation. A number offaint higher-order molecular mass bands may be seen that are notpresent in lane 8, indicating that the ligation was successful. Atransformation of this reaction yielded a small number of bacterialcolonies. Lane 10 is a double digest of a plasmid preparation froma bacterial colony obtained following the ligation reaction. Thisdigestion yielded a single 3.3-kb fragment as in lane 5, as well asa 1.2-kb fragment as in lane 6, indicating that the plasmid con-sisted of the pGFPuv expression vector and included the recom-binant His6-serpin PCR insert. Lane 11 is a double digest of aplasmid preparation from a bacterial colony obtained followingthe ligation reaction. This digestion yielded a single 3.3-kb frag-ment as in lane 5, indicating that the plasmid from this colony ispGFPuv and lacks the recombinant serpin insert. Lane 12 con-tains Directload wide-range DNA marker (Sigma).
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than many other traditional PCR enzymes (e.g. Taq polymerase),reducing the frequency of errors in the amplification product. Adetailed protocol for PCR utilizing Pfu turbo can be obtained fromStratagene. To run the reaction, 100 ng of plasmid DNA, 100 ngof each primer, 5 �l of 10� Pfu buffer mix, dNTPs to a concen-tration of 500 nM each, deionized water to 50 �l, and 1 �l ofenzyme (2.5 U) were mixed in a thin-walled PCR tube. A singlecycle consisted of a denaturing phase at 95 °C for 2 min, anannealing phase at 67 °C for 30 s, and finally an extension phaseat 72 °C for 3 min. This sequence was repeated for 25 cycles. Thefollowing considerations should be taken into account when de-signing a PCR cycle. The polymerase extension time should be atleast 2 min/kb of PCR product. The annealing temperature shouldbe 5 °C lower than the lowest primer Tm. If a thermocycler is usedwithout a heated lid, 30 �l of mineral oil should be laid on top ofeach reaction to prevent evaporation of the reaction mixture. Asthe PCR run time can take 3–4 h, the reactions can be startedduring the laboratory period and the thermocycler can be pro-grammed to hold the reactions at 4 °C or 10 °C overnight. PCRproducts can then be stored for extended periods at �20 °C.
Week 6
Restriction Digestion of the PCR Product and Gene Vector—The presence of a PCR product of the appropriate size (1.2 kb)was verified by E-gel (see Fig. 3). The concentration of PCRproduct may vary greatly so both a relatively large (5 �l) and smallamount (1 �l) of the PCR should be loaded into the gel. Thedesired PCR insert should be the major product visible on the gel.
In preparation for directional insertion of the PCR fragment intothe pGFPuv vector, both the PCR product and vector were cutwith SacI and EcoR1. This digestion yields complementary“sticky” single-stranded ends that facilitate ligation of the insertinto the linearized vector. Restriction digestions using two differ-ent enzymes can be performed simultaneously if both enzymesare active in the same buffer. The results of the restriction diges-tions of the vector and the PCR product were analyzed on anE-gel to ensure that they gave DNA fragments of the expectedlengths. Because the restriction sites are near the end of theamplified insert, the mobility of this PCR product does not changeappreciably upon digestion. Similarly, the digested vector closelyresembled the singly cut vector as only a short DNA piece (lessthan 100 bases) was excised from the vector. This step was usedto ensure that no uncut vector remained and that there were nounintended digestions in the vector and PCR product. Followingthe digestion, the DNA was stored at �20 °C. The single-stranded DNA “sticky” ends are especially sensitive to degrada-tion, thus nuclease free reagents were used during all manipula-tions and care was taken to avoid contamination of the reactions.Finally, a preparation of vector digested with only EcoR1 was alsoproduced in this manner for use as a positive control in thesubsequent ligation reaction.
Week 7
Spin Column Purification of Large DNA Fragments—LargeDNA fragments produced by restriction digests of the PCR prod-uct in the previous week were purified by a spin column protocolto remove contaminants. Large DNA fragments were rapidly (inless than 30 min) recovered in high purity using Qiaquick PCRspin purification columns according to the vendor’s protocol (Qia-gen, Valencia, CA). This step was used to remove enzymes, smallDNA fragments (less then 100 bases) and undesirable buffercomponents. In many cloning schemes, the use of a DNA purifi-cation column may provide a rapid alternative to gel-purificationtechniques.
Treatment of the Vector with Calf Alkaline Intestinal Phospha-tase—After restriction digestion, the vector was treated with calfintestinal alkaline phosphatase (CIAP; Promega, Madison, WI) toprevent self-ligation in the subsequent step. CIAP removes 5�phosphates from DNA. Circular plasmids transform and replicate
much more efficiently than linearized DNA. While the digestion ofthe vector with two different enzymes should prevented ligation ofthe vector alone, the use of CIAP prevents ligation of any singlycut vector, reducing the number of false positives in the subse-quent ligation reactions. An excess of CIAP was used in a 30-minreaction to treat sufficient linearized vector. The vector was thenimmediately purified using a PCR spin column (Qiagen) to removethe CIAP enzyme. Purified vector was stored at �20 °C until thefollowing week. An aliquot of digested vector not treated withCIAP was reserved for use as a control reaction in the ligationstep. EcoRI singly cut vector was also treated with CIAP as acontrol.
Week 8
Reaction Ligation and Transformation—Purified digested PCRinsert and vector (treated with CIAP) were ligated to form the newrecombinant plasmid pMAJILK containing the GFPuv- His6-ser-pin sequence (see Fig. 1). Utilizing a recently introduced quickligation kit (New England Biolabs), the ligation incubation timewas 5 min at room temperature. This allowed the ligation reactionand transformation to be completed in a single laboratory period.A molar ratio of �2:1 insert-to-vector was used in the reaction. Atleast 200 ng of linearized vector should be utilized. Initially, onlyhalf of the ligation product was used in the transformation in casethe transformation was unsuccessful. Unused insert and vectorwere stored at �80 °C for future ligation attempts.
Transformations of the ligation products were done using high-efficiency competent cell preparations with transformation effi-ciencies of at least 106 transformants per microgram of viableplasmid DNA. The transformation reactions were plated on LBagar plates containing the appropriate antibiotic. Multiple agarplates should be used in order to plate the entire ligation reactionvolume. To check the effectiveness of ligation, a sample of ex-pression vector cut only with EcoR1 (no CIAP) was treated withligase and compared with an untreated sample. To check theCIAP reaction, a sample of the EcoRI cut vector treated with CIAPalso was included. As a negative control, a sample containing theCIAP-treated vector and insert but not exposed to ligase was alsotransformed. Finally, positive and negative control transformationreactions as described previously were employed to verify theeffectiveness of the transformation procedure.
Week 9
Restriction Analysis of the Gene Products—Following plating ofthe transformation reaction, a small number of colonies weretypically observed (less than 50). A limited number of colonies(�5) were grown in separate 2-ml cultures containing antibiotic,and a plasmid DNA isolation of each culture was performed. Asdescribed previously, these cultures were inoculated the day priorto the laboratory meeting. A diagnostic restriction digestion usingEcoRI and SacI was performed to assay whether the isolatedplasmids contained the recombinant insert. The digestions werestored at �20 °C until the following week.
Week 10
Agarose Gel of the Diagnostic Restriction Digest—The diag-nostic restriction digestions were visualized on an E-gel (see Fig.3). Digestion of plasmids without the recombinant insert yieldedonly linearized vector. Digestion of the desired recombinant plas-mids from recombinant colonies resulted in the obsevation of aliberated insert (1.2 kb) containing the His6-serpin gene as well asthe linearized vector. Utilizing this procedure, our students con-sistently observed insert in �50% of the plasmid preparations.While the observation of an insert in these diagnostic restrictiondigests strongly indicates successful completion of the subclon-ing process, plasmids with inserts should be investigated furtherby sequencing.
Design and Ordering of Sequencing Primers—Primers for se-
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quencing were 22–25 bases in length and were completely com-plementary to the gene of interest. Selection of the site on thegene for the design of primers was based on several factors. First,the spacing of primers was approximately every 400 bases toenable complete and accurate sequencing of the entire sequenceof interest. Second, the melting temperature was 55–60 °C, witha GC content of 45–60% in accordance with the requirements ofthe sequencing facility. Third, primers were checked for self-priming and loops using software from Integrated DNA Technol-ogies (www.IDTDNA.com). Finally, the primers were located �50bases upstream (3�) of the beginning of the sequence to be read.For some commonly used plasmids, commercially prepared se-quencing primers are available.
Week 11
Sequencing of the Plasmids Containing the DNA Insert—Acrucial test of a successful cloning experiment is the sequencingof the gene insert along with flanking sections of the vector.Although automated sequencers are generally too expensive forsmall, liberal arts colleges, DNA sequences can be easily andinexpensively obtained through outside vendors. These vendorstypically require that they be sent purified plasmid and sequenc-ing primer. This process typically requires less than 10–12 �l ofspin-column-purified plasmid sample with a concentration of 150–200 ng/�l. A typical sequencing reaction yields about 700 bases ofreadable sequences and costs $9–13 per reaction, depending onthe company. We used Genegateway (www.genegateway.com),which cost $9 per reaction and had a very short turn-around time(less than 3 days).
Week 12
Interpretation of the Plasmid Sequencing Results—The se-quence was transmitted from Genegateway by E-mail in a com-pressed file which, when expanded, gave both the original se-quence chromatogram and a sequence file. An examplechromatogram is shown in Fig. 4. In this method of automatedsequencing, each terminal base is labeled with a different fluo-rescent dye [15]. As the different DNA fragments migrate througha matrix and pass a detector, the observed relative fluorescentintensities are used to determine the identity of the terminal DNAbase. The automated sequence read can be aligned with theexpected vector/gene sequence using a program such as Clust-alW. There are many ClustalW sites on the web, such as pir.geor-getown.edu/pirwww/search/multaln.html. Discrepancies be-tween the expected and obtained DNA sequences must beinvestigated further. Because there can be errors in the auto-mated reading of bases, it is highly recommended that studentsedit the chromatogram where discrepancies occur to ensure thatthe sequence read correctly corresponds with the chromatogram.Special attention should be paid to the regions of the primers
because error in primer synthesis can result in unexpected mu-tations, and did so in one of our selected clones.
DISCUSSION
Using the experimental procedures as outlined above,all four students in the semester-long laboratory courseindependently produced plasmids containing the GFP-His6-serpin chimera. This experimental series exposed thestudents to a number of modern techniques in molecularbiology and allowed students to utilize these techniques inpursuit of a concrete project-based experimental goal.Students and faculty conferred as a group at the beginningand end of class periods. At the beginning of the semester,these meetings were used to check student calculationsfor the buffer preparation, to discuss time managementduring the remainder of the period, to comment on theday’s protocols, and to introduce the theory underlying theprotocols. Later in the semester, basic protocols (e.g.those supplied by vendors), computational tools, and web-sites were given to students and they were expected todesign their experiments based solely on the vendor in-serts; this forced the students to become more comforta-ble with protocols and experimental time management. Inaddition, students were expected to bring their interpreta-tion of their results to these meetings for discussion. Stu-dents became more independent in their work as the se-mester progressed.
This project continued in the following summer by twostudents and in the fall semester as an independent study.During these periods, the students have been able toexpress and purify the protein using a nickel affinity col-umn to greater than 95% homogeneity from the bacterialexpression system. The chimeric GFP-serpin product wasdemonstrated to have retained its serine protease inhibi-tory activity. As an additional educational benefit, the pro-gress resulting from the work done in this laboratory se-quence and summer session has generated theopportunity for student presentation at scientific confer-ences [16].
Furthermore, using the experimental procedures as out-lined above, the four students participating in the inde-pendent project of Dr. Deibel were able to produce a set ofplasmids containing the His6-N-lobe transferrin chimera.Students in this project conducted 6 h of research perweek on days determined by their individual schedules. As
FIG. 4. Chromatogram from an automated DNA fluorescent sequencing run. Shown is for the His6-N-lobe transferrin plasmidchromatagram produced by automated flourescent sequencing (Genegateway). The chromatogram is viewed using chromas, whichcan be downloaded free of charge at www.technelysium.com.au/chromas14x.html. The automated read of the chromatagram isdisplayed.
180 BAMBED, Vol. 32, No. 3, pp. 173–182, 2004
a result, students needed to coordinate their research withone another, as the specific procedure they needed to runon any given day depended on what other students hadcompleted the day before. During this project, studentsnot only were exposed to “hands-on” research in modernmolecular biological/biochemical techniques, but alsowere able to participate in a group research project.
National organizations (e.g. PKAL), and private and gov-ernmental funding agencies (e.g. Howard Hughes MedicalInstitute, National Science Foundation) have challengedthe undergraduate science community to begin educatingstudents in ways that better model the actual process ofscience. This laboratory series was designed to addressthese recommendations. Some of the potential advan-tages of a semester-long, research-based project are: 1) acloser resemblance to a professional research environ-ment as might be encountered in graduate school; 2)experience with experimental design; 3) an understandingof how different techniques can be integrated to accom-plish a larger goal; 4) and a sense of personal involvementin a “real world” project that may lead to the advancementof knowledge and might be presented to the larger scien-tific community. Although this laboratory series was notstrictly inquiry-based because a particular outcome wasexpected, the series does model the typical experiencesinvolved in a research project, including the opportunitiesto trouble shoot steps that do not work. Such experiencesestablish technical and theoretical foundations on whichstudents can then begin to do independent, inquiry-basedwork.
PKAL asserts that an important aspect of an undergrad-uate’s success in a research environment is feeling thepresence of a supportive community. To achieve this goaland because our students had different backgrounds inlaboratory techniques, we encouraged them to rely heavilyon each other, in addition to faculty. By being attentive tostudent successes and struggles, we were able to pro-mote progressive independence in the laboratory. For in-stance, at the outset of the semester, protocols (e.g. fortransformation) were rewritten and expanded by facultyfrom those supplied by the manufacturer in order to pro-vide students with maximal instruction and explanation oftheory. In some cases, presentations can be given usingexamples taken from educational biological textbooks toexplain scientific principals involved in the protocol [17–21]. As the students came to understand the theory andwhich steps were critical, faculty provided abbreviatedprotocols and then monitored and questioned studentsclosely during the laboratory to ensure they had a clearunderstanding of the protocol and how to carry it out. Atthe end of the semester, the students were provided withthe protocols from the suppliers and expected to expandthem into a more detailed one in their own notebooks.Consultation with faculty and other students was used toensure that the students were interpreting the materialsappropriately. This judicious withdrawal of support forcedthe students to pay attention to the overall goals of aprotocol, as well as important details such as tempera-tures, incubation times, buffer compositions, and ex-pected yields. At the end of the laboratory course, stu-dents were asked to give short detailed presentations on
steps in the protocol, such as ligation or PCR, to reinforcetheir understanding of the principles involved in the per-formed laboratory procedures.
In addition to being representative of the professionalresearch experience, the laboratory series modeled theimportance of good laboratory record keeping, controlexperiments, and good experimental design. Although thetimeline provided in Table I is representative of an idealexperimental series, the instructor must allow for experi-mental setbacks and be flexible in his/her guidance of thestudents over the course of the semester. Examples ofexperimental setbacks experienced by some of our stu-dents included failure to obtain purified DNA and failure toobtain colonies with the transformation following plasmidligation. However, these occasional setbacks providedvaluable educational opportunities. The sequential natureof the experimental series allowed for students to thinkabout what might have gone wrong, adjust their experi-mental design, and then try the experiment again utilizingmaterial from the previous step. Thus, both the instructorand the students should be aware of the amount of mate-rial (e.g. plasmid, PCR product) produced at each stepwhile designing experiments, and save unused material incase problems arise in subsequent experiments. In somecases, materials such as plasmid preparations can beshared among students if one or more are unsuccessful inharvesting plasmid. In other cases, especially later in thesemester when the students are more familiar with theexperimental techniques, students can be encouraged topursue independently whatever experiments are neededto complete the project.
This project-based learning format was well received bythe students. In general, at the end of the semester, ourstudents became very enthusiastic about completing theirproject and in several cases wanted to work on the projectoutside of normal laboratory hours. Student evaluationsrevealed that they considered the “hands on experiencewith biochemical techniques” and that “students were al-lowed to do much of the planning (and) experimental pro-cedure” as positive aspects of the laboratory course. Ad-ditionally, all of the students indicated that this experienceincreased their interest in the subject. Finally, the plasmidconstructs generated in the course have provided startingpoints for additional research projects for several studentsand likely will be featured in further presentations andpublications. Thus, this experience appears to have pro-vided a unique and valuable preparation for a career inexperimental science.
The experimental sequence can be used to advancestudent or faculty research in a wide variety of situations.Such an approach may be especially beneficial for facultyat undergraduate institutions where the focus is on teach-ing and traditional research time is limited. Advances inDNA and protein technology will have an increasingly largesocietal impact. Accordingly, increased emphasis is cur-rently being placed on the role of liberal arts colleges inintroducing their students to relevant research projects.Fortuitously, the availability of new economical molecularbiology kits and reagents is making the use of these tech-niques increasingly feasible in the undergraduate labora-tory. The size of the laboratory section is certainly a con-
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sideration for such an open-ended approach. Our group,being unusually small, allowed for a great deal of individualattention and guidance. However, we are confident thatsuch an approach could be used with a larger group ofstudents. Peer-peer interactions were a significant re-source, even among the group of four. Teaching assis-tants, which we did not have, could also provide a greatdeal of guidance and consultation and facilitate the use ofthis laboratory sequence with larger groups of students.
Because this series was successful, we feel there areseveral options for future Advanced Cell Physiology labo-ratories using this experimental sequence. One is to repeatthe series with a different DNA amplification product asoutlined in the preceding paragraph. As this procedure canbe used with any sequence, a specific set of tagged genessuch as those in a particular signal transduction pathwaycould be produced in different laboratory sections. Alter-natively, a series of homologous tagged genes from ofnumber of species could be produced. The recent com-pletion of a number of genome projects has greatly in-creased the number of potential genetic targets. The gen-eration of libraries of related tagged genes has a myriad ofpotential uses in further proteomic investigations of geneproduct function. Thus, the featured laboratory sequencecould be used to substantially augment the research ofundergraduate teaching faculty.
Acknowledgments—We thank Ishan Dillon, Lacey Verkamp,Monica Silver, Kate Ware, Abigail Gay, Kjersti Knox, Lauren Phill-ips, and Zachary Seymour for their enthusiasm, diligence, andperseverance during the course of the semester. The generousdonation of the His6-serpin cDNA by Mike Kanost and HaoboJiang is greatly appreciated. In addition, thanks go to MelissaFoster, William Harvey, the Howard Hughes Medical Institute(HHMI), and the Earlham College Biology and Chemistry Depart-ments, for provisions of materials and equipment used in thecourse of the laboratory sequence. J. A. B. would like to thankHHMI and the members of Earlham College for making an enjoy-able postdoctoral teaching experience possible.
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