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Connecticut College, New London, Connecticut 06320
Submitted February 5, 2002; Revised April 10, 2002; Accepted April 24, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Key Words: education • two-hybrid screen • integrated learning • classroom research
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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This laboratory project was designed to introduce scientific research to a broad range of students and to demonstrate to students the excitement of discovery. In addition, students are taught to design experiments and to analyze data, including the proper use of controls. The design of the project and that of the course were integrated so that the laboratory would enhance student understanding of the course material. Goals specific to cell biology were also identified: upon completion of the course, the learner will have gained the skills necessary to 1) search genomic databases, 2) search and electronically access scientific journal articles, and 3) write a paper in scientific format. These goals were not shared with the students so that the goals would not influence the students' evaluations. The achievement of these goals was assessed by graded events designed to test the goals and through analysis of student evaluations of the project.
This project would work well in a cell biology, molecular biology, or genetics course because the screen encompasses basic cellular processes, including transcription, translation, and DNA replication, and because the instructor chooses the cellular system to which the project is applied. The course is designed for one college semester with a 4-h laboratory period that meets once per week, although this project could be scheduled into a 3-h lab with only a few modifications. The project is appropriate for sophomores to seniors, it spanned 8 weeks of the course, and it included 12 students in each laboratory section.
The project involves a two-hybrid screen in yeast (Fields and Song, 1989). The screen includes many important laboratory techniques, including bacterial and yeast transformation, a cDNA library screen, isolation of plasmid DNA, restriction enzyme digestion, DNA sequencing, and utilization of genomic databases. This project allows students to observe results of cellular processes covered in the course curriculum. For example, coinciding with lectures on transcription, laboratory sessions allow students to observe yeast turning blue due to activated transcription of the lacZ gene. Thus, students experience a visible result of an abstract, molecular event. Throughout the project, students connect topics learned during the semester and often relate topics learned in lecture to the ongoing research in the laboratory. Examinations given in lecture indirectly supported the research project by incorporating problem-based learning questions. Each exam contained questions typical of problems that students might encounter in laboratory research. Because each student pursues his or her own results throughout the 8-week project, students become invested in the outcome of their experiments and are excited to see their results and to analyze their data.
In addition to the many benefits to students in the class, another advantage to screening a library is that the data obtained can be pursued by students as independent study or honors' thesis topics. The yeast two-hybrid screen can be used to search for unknown protein interactors of a protein of interest to the instructor; thus, this research can directly benefit ongoing research at the college or in the faculty member's lab. Students who are interested in pursuing work on a particular protein-protein interaction can continue work as an independent study. The potential topics for research following the screen have almost endless possibilities, from bioinformatics to bench science. For instance, students can pursue questions relating to protein binding in an in vitro system or use immunofluorescence to investigate the subcellular localization of the two proteins.
Finally, the excitement that students experience in performing original research enhances their interest in the course and in science. At the conclusion of the screen, students research the protein that they identified in the screen by accessing the scientific literature. Students then form hypotheses regarding the significance of the protein-protein interaction that they identified and even propose experiments to test their hypotheses. One student commented that he or she liked the "excitement of trying to discover new things."
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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The transcription factor that activates the reporter gene is made up of two distinct fusion proteins that can combine to reconstitute transcriptional activity (Ma and Ptashne, 1988; Figure 1). The cDNA of the bait is fused to the coding sequence of the DNA-binding domain (DBD) of a transcription factor, and the cDNA library is fused to the sequence encoding the transcriptional-activation domain (TAD) of a transcriptional-activating protein. If and when these two hybrid proteins bind (the fish bites the bait), a transcriptional-activating factor is reconstituted, the two proteins bringing together the two essential parts: the TAD and the DBD. This reconstitued factor will activate transcription of only a reporter gene or reporter genes that are under control of that transcription factor (the Gal4 transcription factor and binding sites are used here; Figure 2). The name of the system, two-hybrid, stems from the fact that the bait fused to the DBD and the cDNA fused to the TAD form two hybrid coding sequences and proteins. This system was first described by Fields and Song in 1989 and has been improved over the years to allow more efficient screening of baits (Finley and Brent, 1994; Grossel et al., 1999; Hanes and Brent, 1989; Inouye et al., 1997; Vidal et al., 1996a). Detailed protocols for yeast two-hybrid screening are available in several manuals (Ausubel et al., 1999; Burke et al., 2000).
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200 ade2-101 gal4 gal80 can1R
cyh2R Gal1::HIS3@LYS2 GAL1::lacZ SPEX4::URA3) containing
plasmid pGalcdk6 was used for this screen and is available upon request. The
two-hybrid screen is also available as a kit from several companies, including
Invitrogen (ProQuest series 10835).
The Laboratory Schedule
The 8 weeks of this project consist of 6 weeks of laboratory work and 2
weeks of DNA sequencing and computer database searching. In the first week of
the project, yeast that retain the bait plasmid are transformed with the cDNA
library plasmid. Each plasmid contains a gene involved in the production of an
amino acid (Figure 2B), so that
a process similar to antibiotic selection in bacteria can be used in selecting
the yeast that were successfully transformed. After selection for yeast that
contain the cDNA library, the screen is performed. Yeast colonies are
transferred by a simple method, called replica-plating, onto medium that
selects for yeast that contain a positive protein-protein interaction. The
primary screen tests for transcriptional activation of the HIS3
reporter gene as determined by the ability to grow on medium lacking histidine
and containing 3-amino-triazole (3AT), which increases the stringency compared
with that of medium simply lacking histidine (which thus requires the yeast to
produce more histidine in order to survive). In the second week, potential
positive yeast (those that test positive in the first round and contain
potential interactors) are retested for growth on plates lacking histidine and
tested for expression of a second reporter gene, lacZ. In Vidal's
system (Vidal et al.,
1996a,
1996b, 1998), both reporter
genes (HIS3 and lacZ) are under control of binding sites for
the Gal4 transcription factor (Figures
2A and
3). Expression of the
lacZ gene is indicated by blue color formation in yeast in a
ß-galactosidase assay (Figure
3). Controls are necessary to ensure reliable results of these
assays. In our screen, three controls are used: a strong positive (Fos/Jun), a
weak positive (pRB/E2F), and a negative control (empty vector). (See Appendix,
Experimental Details and Protocols, for details.) One strength of this
particular yeast system with two reporters is that it decreases the likelihood
of identifying false-positives (Vidal
et al., 1996b; Gibco kit). All hits isolated thus far in
this screen appear to be true cdk6 interactors, but as with all two-hybrid
hits, interactors need to be confirmed by in vitro binding studies. Controls
for false-positives due to nonspecific activation of reporters (e.g., by the
bait alone) are not included in our lab, but they can be included or made part
of independent studies by students who want to continue research after the
project is completed.
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It is important to note that not every student isolates a hit, but after the initial screen, the class works as a group and at each step focuses its labors on the positive outcomes that were isolated by some members of the class. The number of clones tested (yeast cells transformed) varies from 3,000 to 12,000 per student. The number of hits is directly related to the number of clones tested and varies with the bait used. In the third year of this screen, with 43 students, 190,000 total clones were screened, five independent hits were isolated, some isolated more than once. Two hits were known interactors, cyclin D3 and p19, and two were novel interactors. One hit that did not match a known gene in the databases was also isolated; all others were mapped to known open reading frames. At least one potential hit was lost at some point throughout the DNA-isolation procedure. To rescue lost hits, the instructor can intervene during or after the project.
In the third week, using only the yeast that tested positive for interactions, the class isolated the plasmids from the yeast chromosomes and yeast proteins. The procedure used to purify DNA from yeast is called a "smash and grab" and is conducted during two lab sessions (weeks 3 and 4). In essence, a smash and grab is a plasmid prep in yeast. The yeast cells are lysed, the proteins are denatured, and plasmid and genomic DNA is isolated (Ausubel et al., 1999; Sambrook et al., 1989). This DNA is then transformed into competent bacteria (Sambrook et al., 1989). Bacterial transformation allows the plasmids to be amplified for restriction endonuclease analysis and sequencing. Although either of the two plasmids (bait or prey) will transform bacteria equally well, the yeast can be enriched for the cDNA library plasmid prior to the smash and grab by using auxotrophic selection in the yeast culture medium. Once transformed, bacteria replicate the plasmids, which provides sufficient quantity to allow sequencing of the cDNA inserts. In week 5, the plasmid DNA is purified by using Qiagen columns (Qiagen Inc., Valencia, CA) and digested to confirm the presence of a cDNA insert. In week 6, the DNA is sequenced.
In our lab, Pfizer Inc. (Groton, CT) provides us a tour of their sequencing facility, and students learn about high-speed sequencers used in the human genome project. However, a laboratory session devoted to sequencing could easily be added and would likely take 2 weeks to complete. Sending DNA to a university facility to be sequenced is also an inexpensive and fast alternative ($15-$20 per reaction). After sequencing, the student has the sequence of the unknown cDNA that encodes a protein that interacted with the bait protein to activate transcription in yeast. In the seventh week, a search tool called BLAST (Altschul et al., 1997) is used in an attempt to identify the cDNA clone. The search can be performed on any computer with Internet access and returns a list of genes with sequences similar to the sequence of the cDNA identified by the students. The information returned gives several best matches to the sequence entered (see Ausubel et al., 1999). The sequence of the cDNAs usually allows the student to determine which protein the sequence encodes. Having learned the identity of the interacting protein, students perform literature searches to understand the function of the protein, often making important connections to information they learned previously.
The final week of the project is spent discussing the hits. Our experience indicates that this is best done as a poster session in which students can exchange the knowledge they have gained with their classmates. In this poster session, posters were presented by groups of 3 to 6 students who all shared a common hit. This year's class of 43 students in four lab sections resulted in eight posters representing the four hits that were isolated. The overlap of groups presenting the same hit is helpful to students because individual groups commonly form distinct hypotheses regarding the implication of the bait-prey interaction. The poster session allows students to analyze their work and to connect their findings with knowledge gained in other parts of this course.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Graded events relating to the research project included a laboratory report that was written in the format of a scientific paper, a presentation of a poster at a poster session, and a final paper based on the research project. During the semester, students wrote three lab reports in scientific format. The course instructors graded each report, and detailed feedback was provided to enhance student understanding of the format used. The first two lab reports were complete reports written on separate laboratory exercises. The first was based on a 2-week polymerase chain reaction (PCR) experiment, and the second report described the results of a 2-week cloning experiment. These exercises were chosen in part to provide skills that would assist students in the two-hybrid screen and in part because of their inherent importance in the field. In the first lab report, many students had difficulty writing in a detailed and concise manner and had difficulty distinguishing differences between sections of a research paper (e.g., the Materials and Methods section and the Results section). By the second attempt, students were able to produce reports in proper scientific format. The students' progress was evident in the trend of better grades on the second and third reports. This year, the average grade on the first lab report was a B-, the average grade on the second report had improved to a B+, and this average improved slightly on the final two-hybrid report. The grading on all three reports was consistent, with careful attention paid to inclusion of detail, to concise writing, and to careful analysis of the data. The instructors' impression that the students had learned the scientific format by the second report is consistent with the grade trend. (See Appendix, Sample Two-Hybrid Project Lab Report, for an example.)
Students were also graded on the presentation of their posters. Grading was based on the students' ability to communicate results, to answer questions regarding the controls used, and to understand a biological screen. All students demonstrated excellent understanding of the experimental controls used and their importance to the interpretation of data. The lowest score earned on the poster presentation was a B+, and the class average was an A-, which indicates that students met or exceeded our expectations in understanding the scientific method used in the project, including the proper use of controls. (See Appendix, Sample Two-Hybrid Project Posters, for examples.)
Objectives were directly measured by the evaluation of a final paper. Students wrote a paper on what is known about the function of a protein isolated in the screen and formed a hypothesis of how this function may be affecting the function of the bait protein with which they screened the library. This type of examination incorporates knowledge gained in the class and allows the student to apply his or her creativity and self-expression to the formation of the hypothesis. This paper was comprehensive and challenging and required students to accomplish the goals set out at the beginning of the course. For example, to write the exam, the learner had to perform literature searches, read relevant scientific articles, and summarize the functional properties of the protein. The reference sections of these final exams were analyzed quantitatively and qualitatively. The quantitative analysis indicated that 4 students reported three or fewer primary journal articles, 31 students reported between four and nine articles, and 3 students cited nine or more scientific articles. The qualitative analysis indicated that 33 students referenced articles that were categorized as outstanding. This category required references to be relevant to the paper topic and published in high-impact journals. The remaining 5 students cited articles categorized as excellent, a rating indicating that the articles were relevant but were published in journals with less scientific impact. Because all students completed the examination and because journal searching was a required part of that examination, the results clearly indicated that every student met the goal of searching and accessing scientific journal articles.
The project was also assessed through analysis of student evaluations completed at the end of each of the 3 yr the screen has been conducted. Each year, students completed a general evaluation that assessed interest in the course and the learners' ability to apply the laboratory experience to the course material. In the third year, a more detailed assessment was performed upon completion of the course, and the results are presented here. The evaluations were designed to assess students' perception of the outlined goals. Thirty-eight students completed the evaluations. In the evaluation, students were asked to rate their lab skills before and after the two-hybrid project on a scale of 1 to 5, with 1 being "great" and 5 being "poor." Twelve students rated their skills before the experiment high (1 or 2), whereas 36 students rated their skills after the project high. This finding indicates that 92% of the students who rated their skills less than high now rate them high. In response to a separate question, 15 students rated their interest in scientific research a 1 or a 2 before the two-hybrid project, whereas after the project, 30 students indicated their interest in research at a level of 1 or 2. This finding represents a twofold increase in the number of students interested in scientific research after the two-hybrid yeast screen. Although 5 students indicated that the project did not influence their likelihood of pursuing research, it is possible that these students already knew that they wanted to pursue research.
Data from several questions regarding interest in scientific research, student understanding, and the impact on understanding course material are presented in Table 1. These questions addressed the broad-based goals of the laboratory, including the effect of the project on student interest in the course and in research, and the influence of the project on understanding course material. As shown in Table 1, 18 students (47% of respondents) ranked this laboratory as the best lab they had taken at Connecticut College. This response is remarkable considering that 81% of the students in this course were juniors or seniors. Also, 36 of 38 respondents indicated with a 1 or a 2 that the two-hybrid project influenced their understanding of course material. Thirty-four students indicated with a 1 or a 2 that the lab report contributed to their understanding of the research project.
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Goals that were specific to cell or molecular biology were also assessed in this evaluation, and the results are presented in Table 2. The questions in this section measured an increase in understanding of particular subject areas. The scale ranged from 1 (increased understanding) to 5 (no increased understanding). The evaluation strongly indicated that students believed that they gained a greater understanding of proper use of controls (32 students chose a 1 or a 2) after completing the project. Students also indicated an increase in understanding genomic databases (35 students answered with a 1 or a 2) after completing this project. Although students were less positive about the impact of the project on their literature-searching skills (26 chose a 1 or a 2), several students stated that they already had those skills when they entered the course.
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Open-ended questions were also asked to determine the overall impact of the research project. The students were asked, "What did you like about doing an original research project as part of your lab class?" and "What didn't you like?" Several students agreed on what they did not like about the project, commenting on the "ups and downs" of the project, the long labs, and frustration experienced with the lack of their individual results. A common response to the question of what students liked was the "excitement of trying to discover new things" and "gaining experience and skills," as well as "pride in work." Several students also agreed that they enjoyed working with others on the lab and especially on the poster presentation. One student commented, "It (research) isn't just working in a secluded lab all alone." In response to a question addressing why students liked or disliked the lab, one student commented, "Every part of the lab was related to lecture. We were able to apply what we learned."
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Several of these goals were also assessed through a graded lab report, a poster session, and a final paper. Student performance on these events met the goals and exceeded our expectations. Students at the poster session demonstrated the ability to analyze their results and an understanding of the proper use of controls. Students wrote lab reports in clear, concise language, displaying an excellent understanding of the scientific format. The final paper clearly demonstrated that all students were able to research a scientific topic and produce relevant scientific references while distinguishing the quality of articles produced in the search (see Appendix, Sample Two-Hybrid Project Final Paper, for examples).
Several students intend to submit the findings of their classroom research to an undergraduate research journal. This experience will help students to understand the process of writing and submitting work for peer-reviewed journals and will likely further enhance their interest in research. Students who have participated in research-oriented learning and have continued their studies at the graduate level or have pursued research-oriented work have commented that they were better prepared because of this experience (C. Halsey, S. Sheehan, and D. Krull, personal communication).
From the faculty member's perspective, the weakness of this project is that there is considerable time involved in setting up the experiment. Many plates need to be prepared, and a cDNA library needs to be obtained. These cumbersome tasks could be alleviated if a kit would become available that would provide the cDNA library, selection and reporter plates, competent bacteria for transformation, and perhaps even access to sequencing. Regardless, the bait needs to be cloned into the plasmid with care to ensure a proper reading frame, and it is important to confirm by immunoblot that the protein is made (by Western blotting) before the screen is started. If this project can be of benefit to the faculty member, it is at least not only preparation for the course but also an investment in the faculty member's research program.
This year, the laboratory was concluded with a poster session. This session allowed students not only to present work on their own hit, but also to learn about the other proteins cloned by classmates. Those who pursued the same protein were able to compare references and information that they discovered, while discussions with groups that pursued separate leads allowed students to form a more complete picture of the role that the bait protein may play in the biology of the cell. At the poster session, students were engaged and inquisitive, eager to learn from their colleagues as well as the professors.
In conclusion, this project was highly successful, stimulating student interest in class material and in scientific research. One student stated in her lab report, "This experiment most definitely was successful in pulling all that we had learned in the semester back together. When the experiment was started it was a little unclear how this related to the material of the course, but as the weeks went on it became more and more clear. It was very interesting to be able to take part in this experiment as a way to synthesize a course. I enjoyed learning how research works and I think the fact that it was all hands on helped me grasp and explain the concepts of the course better than I could have from just reading or lecture."
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APPENDIX |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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200
ade2-101 gal4 gal80 can1R cyh2R
Gal1::HIS3@LYS2 GAL1::lacZ SPEX4::URA3) was used for the classroom
two-hybrid screen. HW18 is derived from MaV52 (Vidal et al., 1996)
and was constructed to allow differential screening using multiple baits
(Grossel et al.,
1999). HW18 has three reporter genes—two of which depend on
Gal4 binding sites in their promoters (HIS3 and lacZ) and
one, URA3, dependent on LEXA binding sites in the promoter regions
(Grossel et al.,
1999). Thus, HW18 allows screening of two different proteins or
"baits" fused to two different DBDs (DNA-binding domains), each
bait activating heterologous (Gal4-driven or LEXA-driven) reporter genes. In
the first 2 yr of the classroom two-hybrid screen, the library was screened
with two baits: LEXA-cdk4 and Gal4-cdk6. As a way to make the system more
accessible to undergraduates, the library was screened with only the cdk6 bait
in the third year. Simplifying the system greatly helped with student
comprehension. In strain HW18, the two Gal4-driven promoters (HIS3
and lacZ) each contain five Gal4 binding sites upstream of the start
site of transcription. The Gal4-dependent yeast two-hybrid system is available
as a Gibco kit from Invitrogen (ProQuest series 10835). Yeast strain HW18 was transformed with the Gal4-cdk6 expression plasmid, pGalcdk6. Plasmid pGalcdk6 encodes a fusion protein of the Gal4 DBD in the reading frame with the full-length cDNA of human cdk6. This plasmid also contains the LEU2 marker for auxotrophic selection, an ampicillin resistance gene for selection in bacteria, and an ARS/CEN origin of replication. Immunoblots of transformed HW18 confirmed expression of the cdk6 fusion protein. This transformed yeast strain, HW18/pGalcdk6, retains the plasmid when it is grown under auxotrophic selection (synthetic complete media lacking leucine, SC-L) or stored at -80°C in 15% glycerol.
Yeast Transformation Seven to 14 d prior to transformation, yeast strain HW18/pGalcdk6 was streaked onto SC-L; plate preparation protocol is outlined below. Yeast grow to full-size colonies in about 3 d, are stable on a plate stored at 4°C for 1 to 2 weeks, and can be continually passaged onto fresh SC-L for at least several months. Yeast should be maintained at room temperature (RT) throughout the transformation protocol. The transformation should be monitored by using no DNA as a negative control, and transformation efficiency can be determined by counting colonies grown on a selective plate (SC media lacking leucine and tryptophan, SC-L-T). Proper sterile technique throughout the protocol is necessary to prevent contamination. Preparations of media, plates, and solutions are outlined below. During the screening of the yeast and the isolation of yeast containing potential interactors, students need to attend lab several times a week (for approximately 1 week). This extra time spent in lab is more than made up for in the very short lab session in which students prepare their master plate from their screened yeast. In our experience, students enjoy checking up on their screen and often require help in choosing which yeast contain potential hits.
The following protocols are formulated for 12 students (one lab section in our course):
Pre-lab preparation by the instructor:
Each student:
Yeast that grow on SC-L-T media contain the pGalcdk6 plasmid with the LEU2 marker and a cDNA library plasmid with the TRP1 selectable marker. The next step is to select yeast that may contain a protein-protein interaction between cdk6 and a protein from the library.
Selection for Interactors The yeast are plated onto plates containing 3AT to select for yeast producing large amounts of histidine, which is an indicator of a protein-protein interaction. The number of cells inoculated onto selective plates determines the phenotypic differences between positive yeast and negative yeast. Thus, it is important to transfer a minimal number of yeast cells to the selective plates (see protocol below). The amount of time of incubation of the plate before transfer can also markedly influence the results.
Once potential "hits" (yeast that grow relatively more robustly on SC-L-T-H + 3AT) are identified, they are patched onto a new SC-L-T plate. This plate is commonly called the master plate (see Figure 3A). In addition to any potential hits, this plate is also patched with strong positive, weak positive, and negative control yeast. The strong positive contains the cDNAs of Fos in one plasmid and Jun in the second plasmid; the weak positive contains the cDNAs of Rb and E2F; and the negative control contains pGalcdk6 and an empty TRP1-containing vector (pPC86; available upon request). Depending on the number of hits isolated, students can share master plates because as many as 100 yeast colonies can be streaked onto one plate in a grid. This master plate is then replica-plated onto an SC-L-T + 20 mM 3AT plate and is used to perform an X-Gal assay as described below.
Gal-Lift Protocol or X-Gal Assay
Isolation of Plasmids In this lab, yeast selected that contain potential interactors are lysed and DNA is isolated. To enrich for yeast containing the cDNA library plasmid (TRP1), yeast are first grown in SC-T, which allows the majority of the yeast to lose the pGalcdk6 plasmid.
At this point, bacterial transformation and alkaline lysis plasmid preparation can be done by standard protocols. Diagnostic restriction enzyme digestion can be used to differentiate the cDNA library plasmid from the pGalcdk6 plasmid. Once isolated, the cDNA can be sequenced to determine the protein interactor.
Yeast Media Synthetic Complete (SC) Minus Tryptophan (SC-T) (1 l). In one 2-l flask, mix SC-L-T-H-U reagents:
Autoclave 40 min. Let cool to at least 50°C and add the following before use:
Amino Acid Dropout Mix (-Uracil-His-Leu-Trp). Mix 5 grams each of the following: adenine sulfate, alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. (All are available from Sigma Chemical Co., St. Louis, MO.)
Nutrient Solutions. Filter sterilize; do not autoclave.
SC-L-T. Follow above protocol, replacing 8 ml 100-mM leucine with sterile water.
YPD
Autoclave 40 min. Cool to at least 50°C and add the following before use:
For plates, add 0.1 g NaOH pellets and 20 g agar before autoclaving.
Yeast Plates. Add 20 g agar (DIFCO, DF0140-07) per liter of media. Add agar to media before autoclaving. Most protocols call for agar to be added to 450 ml water in a separate flask and autoclaved separately from the media (made up in a total volume of 475 ml). The contents of the two flasks are mixed after autoclaving. Media must be cooled to 50°C (a water bath set at 50°C works well), before glucose and liquid amino acids are added. Depending on the type of media made, volumes of water used are adjusted to reach a final volume (after addition of glucose and amino acids) of 1 l. It is widely believed that the quality of agar can influence transformation efficiency: DIFCO agar is widely considered the best.
Other Solutions
Z-Buffer (1 l). Use 16.1 g Na2HPO4 7H2O,
5.5 g NaH2PO4H2O, 0.75 g KCl, 0.25 g
MgSO4 7H2O, and 2.7 ml ß-mercaptoethanol. Adjust
to pH 7.0 and bring to 1 l with water.
Sorbitol Reagent (100 ml). Use 18.2 g sorbitol (Sigma Chemical, S7547) and 20 ml 0.5-M EDTA. Adjust to pH 7.5 with NaOH and bring to 100 ml with water.
TE 50/20 (100 ml). Use 5 ml 1-M Tris pH 7.4, 4 ml 0.5-M EDTA, and add water to 100 ml.
Works Cited
Grossel, M., Wang, H., Gadea, B., Yeung, W., and Hinds, P.
(1999). Cdk6 can shorten G1 phase dependent upon the
N-terminal INK4 interaction domain. J. Biol. Chem.
274,29960
-29967.
Vidal, M., Brachmann, R.K., Fattaey, A., Harlow, E., and
Boeke, J. (1996). Reverse two-hybrid and one-hybrid systems to
detect dissociation of protein-protein and DNA-protein interactions.Proc. Natl. Acad. Sci. USA
93,10321
-10326.
SAMPLE TWO-HYBRID PROJECT LAB REPORT
Introduction
The ultimate purpose of this experiment is to find out, through the use of
the yeast two-hybrid system, information regarding the function of the protein
cdk6. To learn more about this protein, an individual must identify, and then
understand, the function of other proteins with which it associates. This is
achieved by using the yeast two-hybrid system to express both the cdk6 protein
and proteins encoded by a cDNA library. If and when cdk6 binds with a specific
protein encoded by a cDNA library, the result will be transcription,
visualized by the turning on of a reporter gene and the subsequent production
of blue yeast. Transcription of a reporter gene depends on the binding of a
functional transcription factor to DNA. The transcription factor prompts the
RNA polymerase to begin transcription of the gene. Following the presence of
blue yeast, indicative that a protein-protein interaction has occurred between
cdk6 and the protein of interest (referred to as protein X), the identity of
protein X will be determined through the process of first sequencing its cDNA
gene by using the Sanger dideoxy method, and then scanning the BLAST database
to find out whether the unknown gene was previously cloned. Finally, if the
identity of protein X is known, its functions and role in the cell cycle can
be researched and looked at in light of the function and role of cdk6, to
ultimately provide more understanding of cdk6.
With this purpose in mind, it is important to realize that this experiment would not be possible without the use of a biological screen, which is beneficial when an individual is testing for specific protein-protein interactions. The idea behind a biological screen is that it allows proteins encoded by a cDNA library to be tested with a target protein in conjunction with a large number of other proteins rather than testing these possible hits individually. This experiment would not be possible if this method was not used because testing these interactions individually would take too long. This method provides the most effective method for finding the "needle" (interactions with cdk6) in the "haystack" (all possible proteins that are encoded by the cDNA library).
The complex events of the eukaryotic cell cycle are regulated by a select number of heterodimeric protein kinases. The regulatory subunits of these kinases are referred to as cyclins, and they have been determined to increase and decrease in phase with the cell cycle (Lodish et al., 2000). The catalytic subunits of cyclins are called cyclin-dependent kinases (cdks) because they have no kinase activity unless they are associated with a cyclin. Regulation of the eukaryotic cell cycle, specifically passage through the cell cycle, is controlled by G1-phase, S-phase, and mitotic cdk complexes. When cells are stimulated to replicate, G1-phase, cdk complexes are expressed, which prepare the cell for the S phase by activating transcription factors that cause expression of enzymes required for DNA synthesis and the genes encoding S-phase cdk complexes (Lodish et al., 2000). The S-phase cdk complexes phosphorylate regulatory sites in the proteins that form DNA pre-replication complexes, whereas mitotic cdk complexes induce chromosome condensation, breakdown of the nuclear envelope, assembly of the mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate, and eventually activate the anaphase-promoting complex (Lodish et al., 2000). Specifically, cdk6 has been the center of much research in part because of its ability to have a role in regulating G0/G1 and G1/S transitions. Cdk6 is important in regulation of the late G1 phase into the S phase because of its ability, in conjunction with cyclin D, to phosphorylate pRB. Phosphorylation of pRB protein is initiated by cdk4-cyclin D and cdk6-cyclin D in mid G1 phase. This initial phosphorylation leads to the generation of cdk2-cyclin E, which further accelerates the phosphorylation of pRB. Once phosphorylated, and thus inactivated, pRB is released from the transcription factor E2F. This transcription factor subsequently binds to nuclear DNA and turns on transcription of S-phase genes that allow replication. Arbogast et al. (1999) investigated the possibility that a loss of activity of cdk6 was implicated in the age-related dysfunction of the cell cycle in its initial phase. Following the conclusion of their research, it was determined that the low cdk6 activity in T lymphocytes from older persons was associated with a defective phosphorylation of the endogenous Rb protein and an increased sequestration of the E2F-1 transcription factor. It is possible that this results in early cell cycle arrest.
Also important in the two-hybrid screen are the specific proteins, which are encoded by a library of genes known as a cDNA library. cDNA stands for complementary DNA, which are DNA copies of mRNA. Throughout, cDNA clones were dealt with, which were made prior to beginning the experiment. cDNA clones lack the noncoding introns present in genomic DNA clones. Consequently, the amino acid sequence of a protein can be determined directly from the nucleotide sequence of its corresponding cDNA. Crucial to the production of a cDNA library is the enzyme reverse transcriptase. After the mRNA is isolated from the tissue of interest, reverse transcriptase is then used to synthesize a strand of DNA complementary to each mRNA molecule. The enzyme works by polymerizing deoxynucleoside triphosphates into a cDNA strand by using the RNA molecule as a template. It is important to note that after going through the process of transcription and translation, one cDNA encodes only one protein. The cDNA library is one of the plasmids present within the yeast colony; the other is Galcdk6. Ideally, one of the proteins encoded by the cDNA library specifically interacts with cdk6. The likelihood of this interaction is purely by chance, but it is helped by the fact that the library was made from RNA from a human brain and thus is likely to contain most proteins. Specifically, the human genome spans 40,000-50,000 genes that code for proteins, and of these 40,000-50,000, between 5000 and 10,000 are expressed in the human brain.
The two-hybrid system that is used is based on the system developed by Vidal et al. (1996) in Ed Harlow's lab. In essence, the two-hybrid system uses transcription as a tool to determine whether a protein-protein interaction occurs between cdk6 and an unknown protein. Under normal cellular conditions, transcription occurs when transcription factors bind DNA, and the transcriptional-activation domain (TAD) causes polymerase to activate gene expression at the transcription initiation site. A transcription factor is made up of two parts: a DNA-binding domain (DBD) and a TAD. Although each factor can act alone, both are necessary to activate a gene. This principle is used in this assay, in which the DBD and the TAD are separated and are brought back together only if cdk6, bound to the DBD, binds to protein X, which is bound to the TAD. If they are brought back together, and the original transcription factor is formed, transcription will occur. Prior to the beginning of the experiment, preliminary steps were done. These steps included cloning the cDNA of cdk6 into the correct plasmid vectors, which allowed them to be expressed in the yeast system. In addition, the cdk6 was made to have a Gal4 DBD. The fusion proteins were tested to ensure that they made the expected-size proteins and could activate the reporter gene only when they were bound to another protein.
In this experiment, the yeast strain used was HW18 + pGalcdk6. The two hybrids were produced by two vectors, one encoding for cdk6 bound to the Gal4 DBD. Here, the cdk6 protein acts as the bait in the system. This plasmid contained a promoter, the DBD, cdk6, a nuclear localization signal, a leucine marker, an ampicillin site, and both a yeast and a bacterial origin of the replication site. The second plasmid codes for protein X, which acts as the prey in the system. Protein X is coded for by the cDNA present in the plasmid, bound to a TAD. The cDNA plasmid was similar except for the cDNA and the TAD fragment in place of the cdk6 and the DBD and except for a tryptophan marker instead of leucine.
The first step in successfully screening the cDNA library is to transform the library into the yeast cells. Because the Gal-cdk6 plasmid was already present in the nucleus of the yeast cell, it was our responsibility to see that the cDNA library plasmid was correctly transformed into the cell. To work with conditions that allowed for the most efficient transformation, we had to capture the yeast at the logarithmic stage of growth, which was determined to be at an optical density 600 nm as close to 0.3 as possible. The process of transformation was carried out by using sterile techniques, and selection was used to pick yeast that contained both plasmids.
A very essential aspect of this system is the proper use of selection. The notion behind this process is allowing for the growth of the yeast that contain plasmids with the specific markers, while getting rid of the yeast that did not transform. There were two different types of selections used for working with the yeast and the Escherichia coli bacteria. Plating the bacteria on a medium containing AMP following bacterial transformation allowed the bacteria that did not transform to die. Conversely, during selection for yeast, a medium was used that lacked specific amino acids necessary for the cell to survive. Thus, the only way for the yeast to survive on the medium is to produce the amino acid. This is possible because the specific amino acids, leucine and tryptophan, are present within the cDNA and Galcdk6 plasmids, respectively. Plating the yeast onto synthetic complete (SC) media lacking tryptophan and leucine allows selection to occur for the yeast that have both plasmids. The selection process was utilized prior to the start of the experiment by plating the yeast onto SC media without leucine so that cells with Galcdk6 plasmids would grow on the plate.
The selection process is used again during replica-plating onto SC-L-T-H + 10 mM 3AT (3-amino-triazole) as a method for testing reporter gene transcription. This selects for yeast that have the desired protein-protein interaction between cdk6 and the unknown protein X because yeast with this interaction produce histidine. The HIS3 gene is located on the genome of the yeast and encodes imidazole glycerol phospate dehydratase, which is an enzyme involved in histidine biosynthesis. This HIS gene is referred to as a reporter gene because its presence indicates that the interaction occurred. By growing on a medium that lacks histidine, only the yeast that are producing histidine, and contain the interaction, remain. The presence of the 3AT is crucial to the selection process because of its function as an inhibitor for the enzyme imidazole glycerol phosphate dehydratase. Consequently, the presence of 3AT serves as a further selection for the yeast with the desired interaction. Only the yeast that are producing a lot of histidine, because of the "strong" interaction, can overcome this inhibitor and survive on the medium. When the concentration of the 3AT was increased, and the potential positives gridded, this selection allowed the yeast that were actually producing histidine to be pinpointed. The amino acid-based selection was used for a final time when positives were resuspended in a medium of SC-T. This enhances for the cDNA plasmid containing tryptophan by increasing the likelihood that the yeast will keep the plasmid and release the cdk6 plasmid.
As stated earlier, this two-hybrid system is centered around the processes of transcription and translation. Although transcription is more important in the overall outcome, translation is necessary for the production of the transcription factors. Transcription and subsequent translation of the two plasmid genes produces both the DBD-Galcdk6 and the TAD-protein X complexes. The presence of a nuclear location signal brings the resulting proteins back into the nucleus of the yeast cell from the cytoplasm where translation occurred. If the desired protein-protein interaction occurs within the nucleus, the resulting transcription factor binds to the promoter region of the reporter gene and enhances the transcription of either lacZ or HIS genes, located on the yeast chromosome. Specifically, the lacz operon produces mRNA that codes for an enzyme ß-galactosidase, which catalyzes the hydrolysis of lactose into galactose and glucose. However, in this experiment, this enzyme reacts with X-Gal in an enzyme substrate reaction to form a blue product that is visible in an assay. In the presence of an alternate substrate, 5-bromo-4-chloro-3-indol-ß-D-galactopyranoside (X-Gal), the product of the enzymatic reaction is 5-bromo-4-chloro-indoxyl, which dimerizes to form a blue product. In essence, ß-galactosidase cleaves the substrate, X-Gal, which thus produces the blue color.
The presence of the blue color indicates that the clones of yeast have given a positive reporter indication for interaction with cdk6. Thus, it is necessary pick out the cDNA plasmid so that the cDNA can be isolated and sequenced. The process of isolation was by means of a smash-and-grab procedure. This was then followed by bacterial transformation, which is a means of separating the two plasmids. Restriction analysis of these plasmids, followed by gel electrophoresis, allowed for the differentiation between cDNA and Galcdk6 plasmids. The cDNA plasmids were then sequenced by using the Sanger method, and the resulting information was used in a BLAST search of protein identities. This knowledge can then be used to make conclusions about the cellular functions of cdk6.
Materials and Methods Yeast Transformation. The morning of lab, HW18 yeast was resuspended in 5 ml sterile water and vortexed to disperse any clumps. Then 50 ml of YPD + Glucose culture was inoculated with yeast to OD600 = 0.130 Å. This culture was grown until OD600 = 0.30 to 0.34. Fifty milliliters of yeast at OD600 = 0.207 was poured into a 50-ml conical tube. This was spun in the tabletop centrifuge at 1800 rpm at 20°C for 4 min. The media (supernatant) was then poured off into a liquid waste container, and the yeast pellet was resuspended in 20 ml of sterile room temperature water. The resuspension was performed using appropriate sterile techniques. After resuspension, the tube containing the yeast pellet was spun at 1800 rpm at 20°C for 4 min. The supernatant was poured off, and the yeast was resuspended in 4.5 ml of freshly prepared TE/LiAc solution. A total of 40 ml was prepared from the stock solutions and contained 4 ml 1-M LiAc, 0.4 ml 1-M Tris, 0.08 ml 0.5-M EDTA (ethylenediamine tetraacetic acid), and 35.52 ml sterile water. The yeast and this solution were spun at 1800 rpm at 20°C for 4 min. The supernatant was poured off, and the yeast was then resuspended in 250 µl of fresh TE/LiAc solution. Four tubes were prepared using the following: 1 µg of 1 µg/µl of cDNA library plasmid DNA, 5 µl of 10-mg/ml denatured single-stranded salmon testes DNA (ssDNA); 100 µl of the yeast prepared above was added and vortexed, and then 600 µl of freshly made PEG (polyethylene-glycol) was added, and the mixture was vortexed again. The PEG solution consisted of 40% PEG, 10 mM Tris, 1 mM EDTA, and 0.1 M LiAc. All four tubes were then incubated at 30°C for 30 min. Seventy microliters of sterile DMSO (dimethyl sulfoxide) was added to each tube, and the tubes were inverted. The cells were then heat-shocked by incubating them at 42°C for exactly 15 min. During the 15 min, the tubes were swirled four times. After the 15 min, the tubes were immediately placed on ice for 2 min. Next, the tubes were spun at 2500 rpm for 4 min at room temperature. The supernatant was removed, the pellet was washed in 0.5 ml sterile water by pipetting up and down, and the tubes were then spun again at 2500 rpm for 4 min. The supernatant was pipetted off carefully. For the cDNA library tubes, each pellet was resuspended in 700 µl of water, and each tube was plated onto two plates of SC-L-T using 350 µl/plate for a total of eight plates. Glass beads were used to spread and allow for even growth by shaking the plate back and forth. The yeast plates were then labeled with name and date, taped together, and placed into the 30°C incubator for 3 d.
The eight SC-L-T plates were removed from the incubator, and seven of the plates were replica-plated and replica-cleaned onto seven SC-L-T-H + 10 mM 3AT plates. These plates were grown at 30°C and were replica-cleaned after 1 d. The eighth plate was placed in a 4°C refrigerator to determine transformation efficiency. The next day, because of overgrowth, the seven plates were replica-plated onto a new set of seven SC-L-T-H + 10 mM 3AT plates.
Gridding Positive Yeast. Forty-eight hours later, after being grown at 30°C, the transformation efficiency was determined by calculating the number of colonies/µg cDNA library. A master plate was made by gridding potential positive colonies from the seven SC-L-T-H + 10 mM 3AT plates onto SC-L-T plates, and the second set of seven SC-L-T-H plates were replica-cleaned again. The plates were incubated at 30°C. Twenty-four hours later, another set of potential positives from the second replica-cleaning were gridded onto SC-L-T plates to create a second master plate. The seven SC-L-T-H + 10 mM 3AT plates were discarded. The two master plates were then replica-plated onto SC-L-T + 20 mM 3AT plates 24 h later. The yeast patches were also lifted onto MagnaGraph nylon membrane (Fisher Scientific). This lifting was performed by first putting a membrane down on the master plate until it was moist; then, once it was removed, the membrane was placed on a YPD plate with the yeast side up and incubated at 30°C overnight. The master plates were stored at 4°C. As a result of difficulties performing the Gal lift by the next day, the plates were removed from the incubator and placed in the 4°C refrigerator 24 h after being placed in the 30°C incubator.
Gal-Lift Protocol. The following was performed for both membranes. On 11/1/01, the Gal lift was performed by first preparing a solution consisting of 2.0 ml Z-Buffer, 12 µl ß-mercaptoethanol, and 150 µl X-Gal (20 mg/ml). Tweezers were used to lift the membrane from the YPD plates, immerse it into liquid nitrogen, and subsequently place it on a paper towel to let it dry/thaw. The mixture described above was put into the lid (upside down) of a petri dish. A Whitman circular filter was placed on top of this liquid, which allowed it to become evenly wet. Once dry, the nitrocellulose membrane was added, yeast-side up, and then covered with the bottom of the petri dish. The plate was wrapped in aluminum foil to prevent it from drying and was placed in the incubator at 37°C for 24 h. The X-Gal was returned to the freezer, and the liquid nitrogen secured. The plates were removed from the incubator 24 h later, and the results checked.
Plasmid DNA Extraction from Yeast Transformants. On 11/7/01, two potential positives were selected from the SC-L-T master plate with a sterilized toothpick, and each was resuspended in 5 ml of SC-T media in a snap-cap tube. A different toothpick was used to scrape each of the two yeast clones and then dropped into the 5 ml of culture. The snap-cap tube was placed in the shaking incubator at 30°C at 250 rpm for 24 h.
After the 24 h, the snap-cap tube was removed from the shaking incubator, and the 5 ml of yeast was spun down in the tabletop centrifuge for 5 min at 2000 rpm. The supernatant was discarded, and the cell pellet was resuspended in 500 µl of sorbitol reagent and transferred into microcentrifuge tubes. Twenty microliters of Zymolyase solution was added to each tube, and the microcentrifuge tubes were placed in the 37°C incubator for 60 min. Next, the microcentrifuge tubes were spun at full speed for 1 min and the supernatant was discarded. The pellet was then resuspended in 500 µl of TE 50/20 solution, 50 µl of 10% SDS was added, and the solution was mixed and then placed in a 65°C incubator for 30 min. The microcentrifuge tubes were removed from the incubator after the 30 min, 200 µl 5-M Kac was added to each solution, and then the tubes were placed on ice for 60 min. The tubes were removed from the ice and spun at full speed for 5 min. The supernatant was then pipetted into a fresh tube. Seven hundred microliters of isopropanol was added to each supernatant and mixed; then, the contents were incubated at room temperature for 5 min.
The microcentrifuge tubes were then spun for exactly 10 s at full speed, the supernatant was poured off, and the pellet was air-dried for 10 min. To help with the drying process, a Kimwipe (Kimberly-Clark Corp.) was used to absorb extra moisture. The DNA pellet was resuspended in 100 µl of TE RNAse at a concentration of 100 µg/ml and then incubated for 30 + min at 37°C. The tubes were then spun for 1 min, and the supernatant was transferred to new, labeled tubes. After spinning, 300 µl NaI solution and then 10 µl of Glassmilk (BIO 101) were added to each tube. The DNA was then allowed to bind to the Glassmilk for 5 min at room temperature while occasionally being vortexed. Next, the tubes were spun at full speed for 2 min, then the Glassmilk/bound DNA pellet was washed three times with 500 µl of New Wash (BIO 101). After the third wash, the Glassmilk pellets were redissolved in 50 µl of TE. The tubes were spun for 1 min, and the supernatant containing the plasmid DNA was saved into a new, labeled tube. The process of transforming 10 µl into 50 µl of competent bacteria was performed and is described below.
Transformation of Competent E. coli with Plasmid DNA. A tube of competent cells was obtained from an ice bucket. The transformations were performed by mixing 10 µl of plasmid DNA samples with 50 µl of competent E. coli cells. The cells were then incubated on ice for 30 min. The cells were then heat-shocked for exactly 90 s by placing the microcentrifuge tubes in a 42°C water bath. After the 90 s, the cells were immediately placed on ice. Eight hundred microliters of LB (Luria Bertani Broth) + Glucose was added to each tube, and the cells were then placed in a shaking incubator at 37°C for 45 min. The cells were next spun at a setting of 7 for 1 min. Then, all but 200 µl of supernatant was removed, and the pellet was resuspended in the remaining LB by pipetting up and down. One hundred eighty microliters of this cell slurry was then pipetted onto an LB/AMP (Ampicillin), labeled plate. Twenty microliters of transformed cells was plated on one LB plate without AMP. With the use of proper sterile techniques, the slurry was evenly spread on the plate with the help of a glass wand and a spinning device. The plates were then placed in the 37°C incubator, upside down, for 24 h. The next day, the plates were removed from the 37°C incubator and placed into the 4°C refrigerator.
Preparation of DNA to Sequence. A bacteria colony was picked with a toothpick and dropped into the 5 ml of BHI (brain-heart infusion)/AMP. The inoculated cultures were then placed into the 37°C shaking incubator. This procedure was performed for us and repeated a number of times for individual tubes.
An E. coli tube containing DL1 sample was obtained, and the procedure was followed for the Qiagen-Prep DNA. A flask of 1% agarose gel + EtBr (Ethidium Bromide) was obtained. This was prepared by mixing 1 g LE agarose with 100 ml of 1X TBE (Tris Borate EDTA) and was then heated in a microwave for 3 min with a Kimwipe plug to prevent moisture loss. When the solution cooled, 3 µl of 10 mg/ml EtBr was added. Because the gel had solidified, it was reheated in the microwave until it turned to liquid. When the gel cooled, it was poured into the electrophoresis box, which contained a plastic stopper and an eight-piece comb. After the gel had solidified, 100 ml 1X TBE, which consisted of 20 ml 5X TBE + 80 ml distilled water, was poured on top and the comb was removed.
A 20-µl total volume enzyme digest was prepared by following six steps: 1) Nine microliters of distilled water, 5 µl plasmid DNA, 2 µl NEB Buffer 3 (New England BioLabs Inc., Beverly, MA), 2 µl 10X BSA (Bovine Serum Albumin), 1 µl Not I, and 1 µl Mlu I were added to a microcentrifuge tube. 2) The mixture was then incubated at 37°C for 60 min in a heating block. 3) Four microliters of 6X loading dye was added to the reaction mixtures, and a prepared sample of 500 µg/ml 1-kb DNA standard was obtained. 4) Twenty microliters of the samples was loaded into the gel wells, the lid was closed, electrodes were attached, and the gel was run at 125 V until the bromophenol blue was halfway down the gel. 5) The gel was then removed and placed on the UV box for visualization. 6) A Polaroid picture was taken.
Results Transformation. Yeast strain HW18 + pGalcdk6 was transformed by using plasmid that contained the cDNA library and a tryptophan marker. The cDNA library was 5000-10,000 genes from the human brain. As a way to enhance for transformation of the cDNA plasmid into the yeast containing the Galcdk6, the yeast containing the Galcdk6 plasmid was grown on a medium of SC-L. The yeast containing the Galcdk6 plasmid was inoculated in a YPD+ Glucose culture, which grew to OD600 = 0.307 in approximately 8 h. Specifically, the yeast was transformed when the optical density reached mid-log phase. The growth of the yeast follows a logarithmic scale, beginning at the lag phase with an OD600 = 0.130 Å, and then followed by logarithmic growth until it eventually levels off
