Recombinant Green Fluorescent Protein Isoforms: Exercises To

In the Laboratory edited by

Concepts in Biochemistry

William M. Scovell Bowling Green State University Bowling Green, OH 43403

Recombinant Green Fluorescent Protein Isoforms: Exercises To Integrate Molecular Biology, Biochemistry, and Biophysical Chemistry Barry W. Hicks* Department of Chemistry, United States Air Force Academy, USAF Academy, CO 80840

Molecular biology, the manipulation of DNA and its products, is a facet of biochemistry that too often does not receive due attention from chemists. The discipline is perceived to exist solely in the realm of biology, and is occasionally ignored in undergraduate biochemistry laboratory curricula. This is unfortunate because strides in molecular biology during the past few decades have allowed biochemists to make significant advances, especially in regard to the relationship between protein structure and function. Among the most significant tools of molecular biology to emerge in the past few years are complimentary DNA molecules (cDNAs) encoding a variety of green fluorescent protein (GFP) isoforms. Fluorescence and phosphorescence are phenomena that still fascinate anyone who enjoyed glow-in-the-dark toys as a child. Why not take advantage of that inherent fascination in the undergraduate laboratory? The GFP from the jellyfish Aequorea victoria offers an opportunity to integrate a range of topics in chemistry by investigating the relationship between protein structure and function using fluorimetry. Various aspects of chemistry can be integrated into this experiment with different emphases, depending upon the instructor. Possible areas of emphasis include DNA replication, RNA synthesis, protein synthesis, protein structure, recombinant DNA technology (molecular biology and biochemistry), the chemistry involved in formation of the fluorophore, the structural properties of fluorophores (organic chemistry), the principles of fluorescence and the excited state (physical chemistry), or the design and operation of a spectrofluorimeter (analytical chemistry). Fluorescence spectroscopy also reinforces and extends the concepts of absorption spectroscopy for undergraduate students majoring in biochemistry. Absorption spectroscopy (UV–vis) is commonly used in the biochemistry laboratory for quantitating protein (1), DNA, or RNA (2), for assaying enzymatic activity with chromogenic substrates (1–3), or for monitoring the eluant from chromatographic columns. However, undergraduates, even those who have previously taken physical or instrumental chemistry courses, often understand these methods poorly. In part, this may be because many discussions about molecular absorption do not discuss the fate of the excited state. Fluorescence spectroscopy is perhaps the best way to reinforce the principles learned in absorption spectroscopy while extending the discussion to the fate of electrons in excited electronic and vibrational states. *Email: [email protected].

The theoretical fundamentals and instrumentation for fluorimetry have been addressed in several past articles in this Journal (4–6 ) and will not be readdressed here. Cloning a plasmid insert in Escherichia coli has also been presented in the Journal (7) and will not be discussed, although experimental detail is provided. The use of fluorimetry as a technique to provide information on protein structure has been addressed before in this Journal (8); however, that report relied upon the weak intrinsic fluorescence of aromatic (principally tryptophan) amino acid side chains, and focused only upon gross structural changes brought about by protein denaturation. Purification of the GFP from A. victoria or recombinant isoforms from Escherichia coli has been carried out at Rutgers University for the past several years (9); it can also be accomplished by HPLC (10) and will not be addressed in detail, although a brief description of our experimental procedure is provided. Instead, this article is designed to provide instructors with the information necessary to use these experiments to help students integrate concepts of molecular biology, biochemistry, and biophysical chemistry using the GFP. Background The GFP was introduced briefly to Journal readers in the January 1997 issue in the “Reports from Other Journals” section (11). Many cnidarians (jellyfish and related marine organisms) utilize GFPs as fluorescent resonance energytransfer (FRET) acceptors for their bioluminescent proteins. In vivo, GFPs fluoresce when excited by luciferaseoxyluciferin or calcium-dependent proteins like aequorin (12). The GFPs are antenna proteins whose function is to act as a fluorophore, but the biological role of these proteins is not known. One could speculate that they have a function in attracting prey or repelling predators. To understand these experiments, the structures of the fluorophore and protein must be examined. The GFP is composed of 238 amino acids and has a mass of about 28 kDa. Crystallography data suggest that the protein exists as a dimer (13). A triad of adjacent amino acids, serine-65, tyrosine-66, and glycine-67 (65SYG67), forms the fluorophore by condensation of the carbonyl group of the serine residue with the amido group of the glycine residue inside the protein. This is followed by oxidation by molecular oxygen to form a double bond to the imidazole-5-one ring, as depicted in Figure 1A. The tyrosine phenolic form of the completed fluorophore is responsible for the UV excitation peak near 395 nm. Deprotonation of the tyrosine hydroxyl group leads to a planar,

JChemEd.chem.wisc.edu • Vol. 76 No. 3 March 1999 • Journal of Chemical Education

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In the Laboratory

highly conjugated, quinonoid resonance structure with an excitation peak in the visible range near 490 nm (13, 14; see also Fig. 3). Mutations can be designed that favor visible excitation, but this can also be promoted by elevating the pH (causing deprotonation of tyrosine-66) on wild-type GFP (15). Although other proteins possess the necessary triad of adjacent amino acids, they do not fluoresce (16 ). This is largely because the tertiary structures of the GFPs uniquely align the three amino acids involved in fluorophore formation in a tight turn that places the glycine amido group within 3 Å of the serine carbonyl group. Furthermore, ab initio calculations suggest that in addition to the close approach of the serine and glycine groups, there is an arginine side chain that hydrogen bonds to the serine carbonyl group to activate the carbonyl carbon for nucleophilic substitution (16 ). Other proteins with the correct amino acid triad lack either the necessary tertiary structure to create the close approach, or the arginine interaction that promotes the condensation reaction. The tertiary structures of wild-type GFP and several mutant isoforms were recently obtained by X-ray crystallography at 1.9 Å resolution (13, 17). Two views of a GFP mutant from the Brookhaven National Laboratory Protein Data Bank at 2.0 Å resolution are depicted in Figure 1B (18). The elements of secondary structure in the GFP include 11 β-strands and one long α -helix. A β-barrel, or β-can, is made from the 11 β-strands that are tightly associated by interstrand hydrogen bonding. The long α-helical segment passes axially through the center of the β-barrel. The three amino acids that form the fluorophore reside near the middle of the α-helical segment. This arrangement creates the necessary tertiary structure to promote fluorophore formation, and it provides exceptional stability to the excited-state molecule. Because the fluorophore is isolated in the protein interior it is relatively resistant to oxidative photobleaching upon excitation. This stability, and the fact that fluorophore formation does not require additional enzymes or cofactors, makes GFP preferable to conventional organic fluorophores for a number of biomedical research applications. The most common use of GFP described in the literature is as a marker for gene expression (19–22). This is accomplished by expressing the GFP under the direction of the promoter region of the gene of interest. Using fluorescent microscopy one can then determine where and when that gene is expressed. A variety of ingenious uses of the GFP demonstrate its versatility and applicability to many areas of biomedical research. Among the more interesting applications of GFP are its use as a probe of membrane potential (23), as a FRET sensor of intracellular calcium ions (24, 25), as a probe to image exocytosis of secretory vesicles (26 ), as a probe in fluorescence recovery after photobleaching (FRAP) experiments for intracellular viscosity (27), as a monitor of individual ATP molecule turnover by single myosin proteins (28), as a fusion protein marker in combination with subunits of the mitochondrial ATP-synthase to examine assembly of the functional complex (29), and as a probe to visualize metastatic patterns of certain cancers (30). Recently, a transgenic mouse that expresses GFP in virtually all tissues (the young mice fluoresce under UV light) was produced and should prove valuable in transplantation studies (31). Fluorescent organic molecules, such as fluorescein or rhodamine derivatives, are available with a variety of fluores410

O N O

N

N

N

O N

O

O-

OH

A

N N

O

O

-

O

O

HN

HN

O OH ser65 tyr66 gly67

N

OH H O

O

HO

H

N

NH O

N

N O

N

O

HO

OH

OH

HO

N

O

HN

HN

HN

+

O

OH

B

Figure 1. A: Formation of the GFP fluorophore requires adjacent serine, tyrosine, and glycine residues. The fluorophore has two excitation peaks that are due to the phenolic and phenolate forms of the tyrosine-66 residue. B: Views of the GFP β-barrel tertiary structure from the side (left) and from the end (right). These were obtained for a BGFP mutant at 2.0 Å resolution from the Brookhaven National Laboratory Protein Data Bank (see ref18) and displayed using RasMol.

A

B

Figure 2. A: Escherichia coli HB101 pGFP streaked onto an amipicillin-containing agar plate, photographed while illuminating only with UV light at 365 nm. B: After expanding GFP transformants, the bacteria were used to write personal notes on new agar (GFP @ USAFA).

Figure 3. Normalized excitation (short wavelengths) and emission (longer wavelengths) spectra of two GFP mutant isoforms, pGFPuv (solid lines) and pEGFP (dashed lines). Excitation spectra were obtained with the emission monochrometer at 510 nm and emission spectra were obtained at the wavelength of maximum excitation.

Journal of Chemical Education • Vol. 76 No. 3 March 1999 • JChemEd.chem.wisc.edu

In the Laboratory Table 1. Amino Acid Mutations in Selected GFP Isoforms and Their Effects on the Wavelengths of Maximum Excitation and Emission λmax ex/ λmax em/ Isoform Relevant Sequencea nm nm Wild-type Phe64 Ser Tyr Gly Val GFP Gln69…Ser72…Phe99…Met153…Val163…Thr203

395

508

EGFP

Leu64 Thr Tyr Gly Val Gln69…Ser72…Phe99…Met153…Val163…Thr203

489

514

GFPuv

Phe64 Ser Tyr Gly Val Gln69…Ser72…Ser99…Thr153…Ala163…Thr203

395

508

BGFP

Phe64 Ser His Gly Val Gln69…Ser72…Phe99…Met153…Val163…Thr203

383

447

YGFP

Leu64 Gly Tyr Gly Leu Gln69…Ala72…Phe99…Met153…Val163…Tyr203

502

527

aChanges

from the wild type are underlined.

cence spectral parameters (32, 33). The spectral parameters of derivatives deviate because of minor structural alterations in the parent structures; for example, an electron-withdrawing group can be added to an aromatic ring. One can also alter properties of the GFP such as the wavelength of maximum excitation, molar extinction coefficient, quantum yield, fluorescence lifetime, and wavelength of maximum emission. In the GFP, however, the alterations are done genetically by engineering cDNAs encoding for different GFP isoforms (34–38). Solvent polarity affects the spectral parameters of any fluorophore (32). Since the fluorophore of the GFP is inside the protein, altering amino acid side chains at or near the fluorophore by genetic engineering may create altered fluorophore “derivatives” or change the polarity of the fluorophore environment. Fifteen amino acid side chains are within 5 Å of the fluorophore and have either been altered already or might be targets for future genetic manipulation (17 ). The cDNAs encoding for many mutant isoforms are already commercially available, including all those listed in Table 1. The mutant GFP isoforms have different spectral properties, which can be related to the protein structure. For example, the EGFP mutant contains two amino acid substitutions near the site of fluorophore formation (17). This has the effect of creating a fluorophore with a red-shifted excitation spectrum relative to the wild-type GFP (by promoting deprotonation of the hydroxyl group on Tyr-66), and a slightly red-shifted emission spectrum (17). DNA shuffling created a second mutant, GFPuv (39). In this isoform three amino acid substitutions that increased the hydrophilic nature of the amino acid side chains in proximity to the fluorophore were made. A third mutation produces a GFP isoform that fluoresces blue upon UV illumination (BGFP) (35), by substituting histidine for tyrosine-66. A yellow-green emitting mutant (YGFP) contains four substitutions, the most important of which is the incorporation of an aromatic tyrosine residue in place of threonine-203 (13). This mutant is significant for two reasons. It has the furthest red-shifted emission maximum published to date and, more importantly, it demonstrates the value of knowing the protein crystal structure in order to rationally predict which cDNA engineering paths to pursue. Ormo et al. knew from the crystal structure that threonine-203 projected toward the tyrosine residue of the fluorophore (13). Putting an aromatic side chain in that position would introduce π electrons that could alter the electronic environment of the fluorophore—and undoubtedly alter

its spectral properties, because the π electrons of aromatic rings are susceptible to polarization. If they interact with the fluorophore excited state, a red-shifted emission spectrum would be anticipated. Other mutations that will produce fluorophores with further red-shifted emission maximum (currently being pursued) can be incorporated into future experiments. Table 1 shows the amino acid sequence data in the wild-type GFP and some mutant isoforms. In addition to the GFP mutants shown in Table 1, other important mutations have been created. One mutant fluoresces better at 37 °C than at 30 °C (for mammalian cell work instead of bacterial cell work) (40); others increase expression in mammalian cells (better folding and solubility) (41), or in plant cells (by removal of a cryptic mRNA splice site) (42). Experimental Procedures The DNA plasmids encoding for the GFP mutants and the polyclonal antibodies were obtained from Clonetech, Inc. The E. coli HB101 strain was from Promega. All chemicals were from Sigma or Aldrich. Sterile, disposable plasticware was purchased from Sigma. Precast electrophoresis gels and cation exchange resin was from Bio Rad. All media were sterilized in an autoclave before use. Bacteria were grown by placing 25 mL of Luria–Bertani (LB) medium into a sterile bottle, adding 20 µL of HB101 cells, and placing the bottle into a 37 °C water bath with house air bubbling through the mixture via a plugged, sterile 10-mL pipet. Cultures were grown in the log phase until the optical density at 600 nm (monitored by removing aliquots every 1–2 h and checking optical density in a Spectronic Genesys 5 UV–vis spectrophotometer) was about 0.5. This provides a cell density of about 0.5–1.0 × 108 cells/mL. The bacteria were harvested by centrifugation, and cells were resuspended in 10 mL of trituration buffer (TB) in order to make them competent. The competent bacteria show higher transfection efficiencies when used immediately after preparation, but they can be frozen in TB containing 10% glycerol (v/v) if necessary. Transformation was done essentially as described in the Promega Protocols catalog (43). Briefly, this consists of adding 20 ng of GFP plasmid DNA (from a 100× stock solution in water) and 3 µ L of DMSO to 200 µL of competent bacteria in TB. After addition, the mixture is kept on ice for 30 min


411

In the Laboratory

to allow the cells to take up the DNA. The cells are then diluted to 2 mL with LB medium and kept at 37 °C for 1 h to allow transformed cells to begin to express proteins necessary for selection. Aliquots of the mixture were plated onto agar in sterile petri dishes and spread with a sterile inoculation loop for ampicillin selection. After 24 h, colonies were identified by exposure to light from a 365-nm hand-held UV lamp. The E. coli HB101 pGFP were grown by inoculating 50 mL of LB medium containing ampicillin, and growing the cells overnight at 37 °C with aeration from the house line. To minimize light scattering, the GFP was freed by lysing the cells in 0.1 M NaCl containing 0.1 % Triton X-100 and sonicating to break up the DNA and cell walls. However, alkaline hydrolysis in 0.2 M NaOH (followed by immediate addition of 1 M Tris pH 7 to restore near-neutral pH) can also be used to liberate intracellular GFP (44 ). The free GFP was purified by precipitation in 67% acetone (the protein is remarkably stable in several organic solvents), size-exclusion chromatography using Sephadex G-75-120, and immunoaffinity chromatography using polyclonal anti-GFP and protein-G Sepharose. (See references 9 and 10 for alternative purification strategies.) The purity of the GFP was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–15% gradient gel. The purified GFP was hydrolyzed in 6 N HCl at 110 °C for 48 h, and the resulting amino acids were separated by ion exchange chromotography using Bio Rad AG 50W-X8 resin (1). The identity of the amino acids in each fraction was then determined by thin-layer chromatography on silica plates using chloroform/methanol/ammonia/water (5:2:1:1 v/v) as the mobile phase (1). Other experiments related to the GFP that were performed in the spring of 1998 included isolating GFP-encoding plasmid DNA from E. coli for transient transfection of eukaryotes (43), and isolating polyA mRNA from GFP-expressing eukaryotic cells by polyT affinity chromatography (a prerequisite for the in vitro translation project below) (1). Although the bacterial cultures can be used to obtain excitation and emission spectra, partially purified GFP fractions reduce light scattering and produce better spectra. At various points throughout the purification, solutions containing GFP were qualitatively analyzed by comparing the emission of different mutant isoforms with 365-nm excitation light from a hand-held UV lamp. Each mutant was also examined alone for emission intensity when excited at both 254 and 365 nm. For quantitative analysis, solutions containing the GFP were transferred to a quartz cuvette and the excitation and emission spectra of the two mutant proteins were obtained in an Aminco 4800C spectrofluorimeter. Excitation spectra were recorded first, followed by emission spectra at the wavelength of maximal excitation. For later discussion, emission spectra were also obtained at excitation wavelengths far from the wavelength of maximal excitation. Results and Discussion All of the plasmids used contain DNA encoding for a GFP isoform, as well as the gene encoding for β-lactamase (to allow selection of transformants by ampicillin resistance). The effects of lactam-containing antibiotics on ribosomal function and the design of new penicillin-derivative antibiotics can be discussed if desired. Time constraints may prevent 412

growing and transforming bacteria in a single laboratory period. Students can grow and freeze cells in one period, or aliquots of frozen competent cells can provided by the instructor for the transformation. After successful transformation, E. coli HB101 pGFP were grown in media, isolated by centrifugation, and lysed to liberate the GFP isoforms. Precipitation and column chromatography were used to purify the GFP, and purity was evaluated by SDS-PAGE. The pure GFP isoforms were analyzed by fluorimetry, and then digested for amino acid analysis using TLC. Figure 2A shows an agar plate streaked with bacteria expressing GFP, illuminated with a 365-nm hand-held UV lamp. The bright green fluorescence is easily visible, and cells expressing different GFP mutants are readily identified by the differences in emission wavelengths or intensities. This “shedding of light” onto the traditional bacterial resistance transformation experiment, by itself, adds a degree of fascination to the conventional experiment and makes it worthwhile. If desired, students can “see their name in lights” by using the expanded colonies to write their name, initials, class year, etc. using fluorescent bacteria as shown in Figure 2B. The Stokes’ shift and the dependence of emission intensity upon excitation wavelength were first qualitatively compared by illuminating the bacterial colonies on agar plates with 254- or 365-nm light from a hand-help UV lamp. This was followed with a quantitative description from the excitation and emission spectra. Figure 3 shows the excitation and emission spectra for two GFP mutants isolated by two different pairs of students. Each pair of students possessed their own unique fluorophore. This personalizes the experiment and increases student motivation. The inevitable comparisons

IC

S2 Ab

ISC

S1 FE

T1 PE

S0 Figure 4. A Jablonski diagram to show the transitions possible for molecular electrons into and out of excited states. The electronic energy levels are indicated as S0 (the ground state), S1 (first excited state), S2 (second excited state), and T1 (the first triplet state). Vibrational energy levels are shown as shorter lines in each electronic energy level. For simplicity, rotational energy levels are shown as the shortest horizontal lines only in the S2 excited state. Radiative absorptions (Ab) are shown as straight dashed lines. Fluorescent emission, FE, and phosphorescent emission, PE, are shown as straight solid lines. Vibrational relaxations (radiationless deactivation) are shown as downward curves. Internal conversion, IC, between vibrational energy levels in different electronic states, and intersystem crossing, ISC, are indicated by horizontal curves.


In the Laboratory

among pairs of students also enhance group learning in the laboratory. The excitation and emission spectra of the GFPuv and EGFP mutants in Figure 3 were obtained using partially purified protein. Although the plasmid manipulations and bacterial transformations were done by students working in pairs, the fluorimetry was done by two groups of six students using samples selected at random. Since a working knowledge of the fluorimeter and its software were not the immediate goals for this class, the instructor obtained spectra after a brief description of the instrumental components to each group. This worked well, but might prove difficult with larger classes, as would any instrumental technique (NMR, EPR, etc.) where instrument costs and space prevent every user from having individual equipment. The effects of the amino acid substitutions on the GFP excitation spectra can be correlated to differences in protein structure near the fluorophore and can provide a sound basis for a general discussion of the excited state, fluorescence, and the relationship between protein structure and function. This discussion may be best introduced with a Jablonski diagram (Fig. 4), which many students may remember from physical chemistry (if that is a required course). Soliciting or providing definitions for key vocabulary such as electronic energy levels, vibrational energy levels, absorption, emission, relaxation, and internal conversion is used to begin a discussion of the excited state. Biochemistry students often have a variety of misconceptions about the excited state, especially if physical chemistry and instrumental analysis are not required courses. This may be a relic of freshman chemistry. While most general chemistry curricula explore atomic absorption and emission, molecular absorption is rarely discussed, so vibrational and rotational energy levels are ignored. Even when physical chemistry is a prerequisite, the differences between a molecular absorption spectrum and a molecular excitation spectrum are poorly understood (45). After the students have a basic understanding of all the transitions possible for electrons in the excited state from the Jablonski diagram, the lifetimes of electrons in vibrational and electronic states can be discussed. Armed with an understanding of the lifetimes and the transitions possible, students can readily explain that relaxation to the ground vibrational energy level within the excited electronic state before radiative emission is part of the reason for the Stokes shift in fluorescence. When students are asked why emission occurs over a range of frequencies, they can use the Jablonski diagram to explain that radiative emission can be to many different excited vibrational levels within the ground electronic state and that this is a second reason for the Stokes shift. Students can use the Jablonski diagram to explain why excitation at wavelengths other than that of maximal excitation could lead to a similar emission spectrum, but with reduced intensity. The Jablonski diagram also gives students an understanding of phosphorescence. Knowing that phosphorescence requires formation of a triplet state, students can see why it is a relatively rare phenomenon and appreciate that the lifetime in the excited state is longer. Lastly, students understand how molecules in solution can absorb radiation without radiative emission: releasing the energy as heat to the solvent is an alternative, through internal conversion to excited vibrational levels within the ground electronic state and subsequent relaxation through vibration, rotation, and collision.

Since a π to π* transition within the conjugated ring system is responsible for the UV excitation (14), an understanding of the fluorophore structure as mentioned earlier is valuable. If the red-shifted mutations are going to be correlated to solvent polarity, an understanding of the protein structure is also necessary. Solutes and solvents can be discussed, and the differential effect of solvent polarity on solute–solvent interaction with the ground and excited state can be introduced. Our discussion concluded with comparisons of the primary sequences of the two mutants and an attempt to rationalize the spectral differences by relating the amino acid substitutions to solvent effects for conventional organic fluorophores. Since fluorophores with electrons in the excited state tend to have greater dipole moments than when in the ground state, the excited state becomes more stable relative to the ground state with increasing solvent polarity and can result in a red-shifted spectrum (32). Mutations in GFP cDNA that encode for amino acids with smaller aliphatic side chains or which replace aliphatic or aromatic side chains with charged or polar side chains increase the dielectric constant near the fluorophore. Such mutations should, by analogy to solvent polarity and organic fluorophores, lead to red-shifted spectra. At a first approximation, the substitution of leucine for phenylalanine in EGFP increases the polarity (46 ) near the fluorophore slightly and should result in a slightly red-shifted emission spectrum compared to the wild-type GFP, as it does (see also Table 1). It also removes an aromatic ring that influences the fluorophore. In reality, besides solvent polarity, other factors must be considered. A complex network of hydrogen bonding between amino acid side chains in the vicinity of the fluorophore affects the degree of Tyr-66 ionization and can alter the excitation spectrum. The proximity and structure of the altered amino acid side chains interacting directly with the excited state of the fluorophore can stabilize the excited state and alter the emission spectrum (see refs 13 and 17 for more detailed analyses). Since several GFP mutants are in the Brookhaven National Laboratory Protein Data Bank (18), this laboratory could also be used as an introduction to protein crystallography (47), and to software like RasMol that can be used to view protein tertiary structures. During the fall semester of 1997, our biochemistry laboratory centered on different aspects of GFP biochemistry (Table 2) to provide students with the basic biochemical techniques indicated. Teaching the underlying biochemical methods and techniques while concentrating upon a single protein provides unification to the laboratories. This prevents Table 2. GFP-Related Laborator y Exercises in FirstSemester Biochemistr y Laboratory

Techniques

Transformation of E. coli with GFP plasmids

Cloning plasmid inserts, antibiotic selection, bacterial culture

Isolation of pure GFP

Size-exclusion and affinity chromatography

Purity of GFP

Protein quantitation, electrophoresis

Amino acid analysis of GFP

Protein digestion; ion exchange and thin layer chromatography

Isolation of GFP plasmid DNA

DNA purification, DNA melting


413

In the Laboratory Table 3. GFP-Related Laborator y Projects for Second-Semester Biochemistr y Project

Techniques

References

Production of polyclonal antisera to GFP

Animal work, western blotting

50, 51

Characterization of scorpion fluorophore

Fluorimetry

52

Transformation of plants with GFP

Plant cell culture, agrobacteria

53–55 56, 57

GFP plasmid DNA and cationic liposomes

Electron paramagnetic resonance

Streamlining E. coli transformation

Molecular biology

41

FRET using GFP

Fluorimetry, molecular biology

23, 24, 58

Eukaryotic transformation with GFP plasmids

Cell culture, fluorescence microscopy 59

GFP peptide mapping and protein modification Fluorimetry, electrophoresis, NMR

60, 61

Site-directed mutagenesis of GFP

PCR, cloning, molecular biology

13

In vitro translation of GFP from mRNA

Electrophoresis, autoradiography

41

Engineering mammalian GFP vectors

Recombinant DNA technology

18–21, 41

laboratory experiments from being perceived as unrelated, a common problem for undergraduates. This unification can be extended to a variety of GFP-related, project-oriented exercises for the second-semester laboratory. Project-oriented exercises increase learning and motivation by giving the student a greater stake in the outcome. They also provide an experience that more closely resembles the atmosphere in a research laboratory (48, 49). For the spring semester of 1998 five pairs of students chose from the 11 GFP-related projects listed in Table 3. Work on the projects occupied the students’ laboratory time for the majority of the semester. The projects emphasized various aspects of molecular biology and biophysical chemistry to different degrees, allowing students to select fields that appealed to their personal interests and preventing overlap on instrumentation. This experiment brings state-of-the-art experimentation to the undergraduate classroom. Undergraduates often become frustrated with “canned” laboratory experiments because they do not see the value in “reinventing a 50-year-old wheel”, especially in capstone senior-level courses where they rightly perceive themselves to be new chemists. The GFP was first cloned in 1992 and did not become commercially available until 1994. However, a recent search of the Medline database produced more than 900 scientific papers using the GFP in the past few years, and that number is sure to grow rapidly in the future. There is also a World Wide Web news group of researchers who commonly use GFP and other fluorescent proteins (62). The Web site provides information that could be used for student assignments. The importance of GFP to a variety of fields in biomedical research is apparent in the high quality of journals that have published articles related to GFP. The article of Chalfie et al. (34 ), which was “highlighted” on the cover of Science in 1994, and several articles in the past year on the covers of BioTechniques (41, 63) demonstrate the appeal of green fluorescence to the scientific community. Not only does the broad and current use of GFP in research allow the instructor to easily emphasize the importance of the laboratory being performed, it engages the students’ attention because they perceive the laboratory exercises as being relevant and applicable. The vast literature on the GFP, much of which is referenced by Clonetech Inc. in a product protocol application note (64) or is available via Internet searches, allows the instructor to integrate the laboratory experiments with literature searches to reinforce the value of the exercises. 414

Equipment (not complete for projects) Autoclave (or filters for sterilizing media) Clinical centrifuge and sterile centrifuge tubes 37 °C water bath, house air line (or air cylinder with regulator, or aquarium pump) or 37 °C shaking incubator for bacterial growth (ambient growth is possible, but slow) Sterile pipets, medium bottles, petri dishes, and inoculation loops UV–vis spectrophotometer (Spectronic 20 will suffice) Hand-held UV lamp or, preferably, a fluorimeter Chromatography columns Electrophoresis apparatus pH meter for buffer preparation Silica TLC plates

Chemicals (not complete for projects) tryptone polyclonal antibodies GFP-encoding DNA plasmids yeast extract NaCl NaOH ampicillin agar CaCl2 MgCl2 CH3COO {Na+ HCl Dimethylsulfoxide (HPLC grade) LB medium: 10 g bacto-tryptone, 5 g bacto-yeast extract, 5 g NaCl in 1 L water, adjusted to pH 7.5 with NaOH and autoclaved in a 2-L flask before use. For expansion of transformed E. coli, 50 µg/mL of ampicillin is added to the cool medium. Agar-LB medium containing 12 g of bacto-agar per liter, autoclaved in a 2-L flask, cooled to 50 °C and 50 µg/mL of ampicillin added. This is poured into sterile petri dishes. TB: 100 mM CaCl2 , 70 mM MgCl2, 40 mM NaOAc adjusted to pH 5.5 with HCl. Tris buffer for size exclusion and ion-exchange chromatography and electrophoresis Sephadex G-75-120 size exclusion gel Polyclonal anti-GFP for immunoaffinity chromatography Protein-G agarose for immunoaffinity chromatography Coomassie G-250 for Bradford Protein Assay Acrylamide or precast gels for electrophoresis Sodium dodecyl sulfate


In the Laboratory Coomassie R for electrophoresis gel stain Methanol for SDS-gel staining/destaining Acetic acid Ion-exchange resin Indicator-free TLC plates Ninhydrin spray reagent for detection of amino acids on TLC plates Methanol, chloroform, ammonia for TLC mobile phase Ninhydrin spray for amino acid detection on TLC plates Triton X-100

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Recombinant Green Fluorescent Protein Isoforms: Exercises To

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