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APPROACH

I.     Systems analysis of Halobacterium sp. will provide a sound basis for developing secondary school supplemental instructional materials that can engage both teachers and students in and familiarize them with current, innovative, multidisciplinary systems biology research.

A.  A model system such as Halobacterium sp. can serve to engage students and teachers with the Archaea, an important but often- at the pre-college level- neglected domain of life.

B.   An integrative computational and experimental teaching method will demonstrate the importance of systems analysis to study regulatory and metabolic networks in biological systems.

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JUSTIFICATION

Rationale for using Halobacterium sp. for an integrated systems biology research and education program. Halobacterium sp. satisfies many criteria that make it an attractive model system for systems-biology research and education.  Specifically:

1.    It is non-pathogenic and easy-to-culture to very high cell densities (>10⁹ cells/ml).

2.    It has a relatively small genome (2.4Mbp) that is completely sequenced  (Ng et al., 2000)

3.    It is a model system for Archaea, a physiologically diverse group of organisms that are predominantly extremophiles and phylogenetically distinct from both bacteria and eukaryotes  (Reeve et al., 1997)

4.    It harbors a rich physiological diversity including organotrophy, phototrophy, arginine fermentation, photo- and chemotaxis  (Ng et al., 2000)

5.    Halobacterium sp. phenotypes, regulated by oxygen concentration and light intensity, are visually attractive with easily observed bright colors and floating cells making them ideal for laboratory and classroom use.

6.    It is easily lysed by re-suspension in low salt buffers, a property highly desirable for large scale and high-throughput RNA and protein preparations.  This property has the added benefit of easy sterilization eliminating the need for expensive and infrequently available equipment such as autoclaves in the high school laboratory.

7.    It is biochemically, genetically and genomically tractable with a range of easily implemented tools including DNA-mediated transformation, saturation mutagenesis systems, shuttle expression vectors, gene knockout systems, microarray analysis and quantitative proteomics  (Baliga and DasSarma, 1999; Baliga and Dassarma, 2000; Baliga et al., 2002; DasSarma and Fleischmann, 1995; Peck et al., 2000)

8.    The high surface negative charge in Halobacterium sp. proteins for adaptation to a high salt environment   makes them ideal for ab initio structure prediction.  The increased accuracy in prediction of structure will facilitate annotation of genes with no previously known function.

9.    A complete network with an exhaustive number of inferred interactions/associations among the NRC-1 proteins has been constructed in the data exploratory tool Cytoscape developed at the ISB   (Shannon et al., 2002)

10. An exploration of halobacterial physiology in hypersaline environments can stimulate students and teachers to re-evaluate the limits of life in environments that until recently were considered uninhabitable.

Purpose of the integrative approach to education. The revolution that is re-shaping biology, forcing biologists to think more systemically and fostering their increased collaboration with other scientists, is virtually absent from the teaching of biology.  In particular, high school biology typically is still taught from ever-larger textbooks that are compendia of taxonomic and other specialized terms.  The discordance between the teaching and practice of the discipline ill prepares students to actually practice biology.  With the advent of genomics during the last 15 years, biology is now pursued as a melding of hypothesis-driven (wherein speculations based on previous observations are tested in a defined framework) and discovery-driven (wherein the experimental design and data exploration leads to discovery of new phenomena) approaches.  This proposal offers an opportunity to produce current, inquiry-driven, standards-based, supplemental educational materials that more accurately reflect this current melding of hypothesis-driven and discovery-driven approaches in biology.

These materials will reflect a systems biology approach, both in the content and in the professional development provided to teachers.  Schools' science and mathematics teachers will be jointly engaged in carrying out the several subprojects proposed and these projects will involve students from a wide range of courses.  While biology students may be focused on culturing Halobacterium sp. and performing the manipulations proposed (see Experimental Plans: III.  A.1. & 2.; III.B.2.), chemistry students will play a more active role in the spectrophotometric analyses (III.B.3).  Computer science and mathematics students will be more deeply engaged in the data modeling and analyses (III.B.1. & 4.).  All subprojects, however, will encourage participation from students (and teachers) with differing expertise.  Through this approach, as the project is piloted and implemented in schools, the schools' faculties and students will immerse themselves in the multi-disciplinary, collaborative inquiries of systems biology.

The educational materials (and their impacts on students' learning) will be evaluated on an ongoing basis as they are developed, piloted, refined, and field-tested more broadly.  Content will be aligned with the National Science Education Standards   (NRC, 1996) and the inquiry methodology will be assessed for adherence to current best practices   (NRC, 2000).  We will develop and refine specific assessment instruments - front end, formative, and summative - for each of the subprojects described in Experimental Plans, III.  The materials and assessment instruments, themselves, as well as the results of various assessments, will be posted on the ISB education web site and made freely available to educators.

Scientists from the ISB (including NB, PE, RB and VN) are currently engaged in an active student-mentoring program with the Ballard High School Biotechnology Academy, a rigorous science preparatory program, familiarizing students with and providing basic background in current systems biology concepts and approaches.  This work reflects ISB's long-term commitment to K-12 education.  The research cum education activity at ISB will therefore continue past the 3-year term of this project ensuring continued dissemination of the educational supplements that will stay abreast with advances in systems biology through close collaboration with local high schools of varying resources.  

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SIGNIFICANCE

Fourth, the development of standards-based, inquiry driven, deeply engaging education materials through close collaboration among scientists, teachers, and students will contribute to an effective and thought provoking curriculum for high school science.  The recent advances in biology will find their way into developing minds, educating them in concepts of new approaches (systems biology) and fundamental paradigms such as the three domains of life and extremophiles.

 Approach     Justification     Significance    Background     Preliminary Results    Experimental Plans   References   Top

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BACKGROUND

Inquiry-based learning.  Inquiry-based learning lies at the heart of science and meaningful science education.  Inquiry emphasizes thoughtful learner-centered questioning, problem solving, and decision-making.  This approach, supported by the National Science Education Standards (NRC, 1996) and the Benchmarks for Science Literacy  (AAAS/Project-2061, 1993) fosters studentsÕ understanding of science as both a personally relevant process and a dynamic body of information and theories.  Inquiry learning approaches support constructivist philosophies Ðthat we all have to build or construct our knowledge from our experiences. The central challenge in classrooms is helping each student build on her/his previous experiences so as to construct that knowledge base mandated by State or National standards that outline explicit expectations for all students' achievements.

To assist in this Herculean task, exemplary, standards-based, inquiry-driven educational materials build on current psychological understandings of learning processes  (Bransford et al., 2000).  One such scaffold, developed by the Biological Sciences Curriculum Study is the so-called five-E model.  The five E's refer to the fact that the sequence in which concepts, facts and experiences are presented to students is of foremost importance. First, teachers engage students in thinking about a concept through a notable event, discrepant experience, or relevant question. Then students explore the event or question through flexibly structured activities.  Next, the teacher explains, clarifying the concept and defining relevant vocabulary. Then students elaborate and build on their understanding, applying the concept to new situations.  Finally, students carry out activities that enable them and the teacher to evaluate their understanding of the concept. Ideally, the process engages students in investigating further, related concepts.

The educational inquiry materials to be developed through this project align with this process, using and building on such a scaffold.  Cohesive sets of learning experiences include activities of engagement and exploration (Exptal. Plans, III.A.) as well as explanatory, elaborative, and evaluative components (Exptal Plans, III B.).

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PRELIMINARY RESULTS

4. K-12 Science Education.  Over the past decade, we have fostered the transformation of regional K-12 science education.  In 1996 we initiated the Partnership for Inquiry-Based Science, for all of Seattle School District's elementary (K-5) programs -1,200 teachers and 23,000 students. This program has been extraordinarily successful, winning the 2001 KCTS Golden Apple Program Award, and has evolved into a model for the nation.  Shortly thereafter, we set up a Family Science program that has subsequently evolved into an extensive five-year community engagement endeavor, complementing and supplementing the school-based systemic change initiatives.

In both 2000 and 2001, students from five Seattle elementary schools that had been participating in the Local Systemic Change (LSC) program for at least two years outscored their peers district-wide in all categories -enhanced multiple choice, open-ended, and performance assessments -on the nationally administered PASS (Partnership for the Assessment of Standards-based Science) science assessment that are aligned with both the National Science Education Standards  (NRC, 1996) and Benchmarks for Science Literacy   .  Analogous LSC projects at other sites have yielded similar results  (Jorgenson and Vanosdall, 2002; Klentschy et al., 2002; Raghavan et al., 2001).  Of particular note is that these impacts are often most striking among students from disadvantaged backgrounds.

Encouraged by the elementary program's success, we initiated and led the NSF-funded Middle School Science Systemic Change Partnership for grades 6-8 with Seattle and four other King County school districts (Bellevue, Highline, Northshore, and Shoreline).  Like its elementary predecessor, the middle school LSC is now acknowledged as among the top school science programs in the country. Representatives from both programs have been invited to present their approaches in national conferences.  A key component for each of these integrated and articulated programs is ensuring long-term sustainability, so a critical element for success is establishing stable, extended funding partnerships.

For the past several years, we also have been actively planning and building the infrastructure for systemic changes in regional high school science, working in conjunction with these same school districts and community partners.  Inauguration of this effort is underway and full-scale implementation awaits receipt of major funding.

We have extensive experience in deriving secondary school instructional materials from cutting edge scientific research.  Pat Ehrman, as a high school biology teacher working through a Murdock Trust fellowship, was instrumental in creating the nationally disseminated High School Human Genome Program (http://hshgp.genome.washington.edu/) that has engaged hundreds of high school teachers and thousands of their students in performing DNA sequencing investigations, contributing their data to the Human Genome database and analyzing their own and others' results.

5. Halobacterium sp. interactive Boolean network.  We have designed a preliminary interactive network simulation model for purple membrane biogenesis.  Once developed and tested this model will be used as a framework for building complex regulatory Boolean networks as proposed in the experimental plans II.A.3 of this proposal as well as for education purposes.  The reviewers can access this preliminary interactive model at this website: http://www.sewardpark.net:8080/halo.  (Password: rhodopsin).

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EXPERIMENTAL PLANS

III. Systems analysis of Halobacterium sp. will provide a sound basis for developing high school supplemental instructional materials that can engage both teachers and students in and familiarize them with current, innovative, multidisciplinary systems biology research. We will engage a few exemplary teachers and students in extended laboratory experiences during the years 2 and 3 at ISB to develop these materials for classroom use.  This approach will provide in-school leadership to promote thoughtful dialogs as students present results of related investigations that reflect different perspectives, approaches, backgrounds, and methodologies.  These leaders will create forums at their schools that model the interdisciplinary synthesis they have experienced at ISB.

A.  An inquiry based approach will familiarize students and teachers with Archaea through a model system such as Halobacterium sp., an important but often -at the pre-college level- neglected domain of life.

Subproject 1.  Design standards-based, inquiry-provoking laboratory learning experiences to provoke an interest for Halobacterium sp. in both students and teachers.  The growth of Halobacterium sp. in high salt can be used to demonstrate the extremes in which life can exist.  For example, students will be provided with 275g of salts and 700mls of water for Halobacterium sp. growth medium and in contrast only 5g of salts for the same volume of water for E. coli. growth medium.  Next, cell pellets for the two different organisms will be re-suspended in high- and low-salt growth media to demonstrate dramatic lysis of the cells in the medium of unfavorable osmolarity.

Subproject 2.  Design a module for investigation of gas vesicles. Halobacterium sp. cultures, normally opaque due to gas vesicle-mediated light diffraction, can be dramatically turned clear when subjected to transient high-pressure by striking a rubber-stopper on the flask.  Students will observe phase-bright gas vesicles responsible for this phenomenon by viewing Halobacterium sp. cells under a phase microscope.  Students will isolate intact gas vesicles by lysing cells rapidly in low-salt medium in the presence of DNase and then centrifuging the lysate at low speed, and will explore properties of the vesicles. Students will manipulate environmental conditions such as light intensity and oxygen tension to investigate why and under what conditions gas vesicles are produced.

Subproject 3.  Develop a module using Halobacterium sp. engineered and spontaneous mutants to illustrate the power of genetics.  Students will culture and study phenotypically distinct Halobacterium sp knockouts (e.g., bat-, bop-).  They will then transform plasmids containing known intact genes into these mutants, determining unambiguously what DNA sequence corresponds to the "knocked-out" gene and confirming DNA as the key genetic material (see III.B.2 for specific details).  Halobacterium sp. contains 91 insertion sequence elements ("jumping genes") that frequently insert into genes for purple membrane biogenesis giving rise to a range of phenotypes. Students will culture wild-type Halobacterium sp. and seek to identify spontaneous phenotypically distinct purple membrane biogenesis mutants in order to perform similar analyses.

B.   An integrative computational and experimental teaching method will demonstrate the importance of systems analysis to study regulatory and metabolic networks in biological systems.

Subproject 1.  Build a simple computational model describing the three biomodules for bacteriorhodopsin synthesis.  We will involve high school teachers and students in refining the simple interactive computer simulation (see preliminary results) for the bacteriorhodopsin synthesis described in Fig. 8.  The simulation will allow students to introduce perturbations at various levels of information and correlate the effect of the perturbation to the output color phenotype.  For example, a bat gene knockout will result in a white phenotype (due to a shutdown in carotenoid as well as Bop synthesis) whereas a bop knockout will result in an orange phenotype (due to exclusive production of bacterioruberins).  Similarly perturbations in environmental factors will also be built into the model so the student can correlate perturbations in the environmental factors to the color phenotype; for example, low oxygen tension induces purple membrane synthesis.  Student learning will be evaluated by posing puzzles that they can first solve in silico and then verify experimentally as described below.  We have already made significant progress by building a proto-type of this simulation model which has aided in identifying the programming logic, the graphic design and user interface. 

Subproject 2.  Develop a kit for conducting simple experiments in the high school laboratory to complement the computational module.  We have four characterized strains of Halobacterium sp. that can be used to demonstrate the effect of perturbations in key elements in the three modules for bR synthesis.  Specifically, the wild-type strain has a pink color phenotype, whereas a strain genetically perturbed to overproduce Bat (bat+), the transcriptional regulator, has a bright purple phenotype. Two stains derived from the bat+ strain are white and bright orange due to insertion knockouts in bat (bat-) and bop (bop-) respectively (Fig. 11). Students can test their predictions from the computational module by complementing the mutants with functional genes.  In the process they will also get a hands-on introduction to the fundamental paradigm that DNA is the hereditary genetic material.  To ensure easy dissemination we will design a kit containing the simulation model on a CD-ROM (with links to additional web-based resources and support), lyophilized stocks for all strains; dried plasmid DNA with the bop and bat genes for genetic complementation; powdered culture medium; DNA transformation reagents and culture plates.

 Subproject 3.  Spectrum analysis of lysates of Halobacterium sp. strains mutated at the various levels in bacteriorhodopsin synthesis.  Since the pigment composition in the various bR mutants vary characteristically, spectrum analysis on the cell lysates can be used to demonstrate the importance of measuring metabolites as an additional level of information.  The experiment will be designed to illustrate the basic principles of spectrophotometry such as Beer-Lamber's law.

Subproject 4.  Involving students and teachers with analysis of large amounts of data from the systems analysis proposed in hypotheses I and II using Cytoscape.  Once the teachers and students are familiar with the Halobacterium sp. model system and the concepts of systems analysis they will be participate in the ongoing research program to stimulate further inquiry-based learning.  They will be given access to experimental and computing resources and data (with appropriate guidance) to address and answer their research questions regarding Halobacterium sp. biology.  

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  References

 Bransford, J.D., Brown, A.L., and Cocking, R.R., (eds) (2000) How people learn: Brain, Mind, Experience,, and School. Washington D.C.: National Academy Press.

Jorgenson, O., and Vanosdall, R. (2002) The Death of Science?  What We Risk in Our Rush Toward Standardized Testing and the Three R's. Phi Delta Kappan 83: 601-605.

Klentschy, M., Garrison, L., and Ameral, O. (2002) Valle Imperial Project in Science (VIPS): Four-Year Comparison of Student Achievement Data. Journal of Research in Science Teaching in press.

NRC (1996) National Science Education Standards. Washington D.C.: National Academy Press.

NRC (2000) Inquiry and the National Science Education Standards. Washington, DC: National Academy Press.

      Raghavan, K., Cohen-Regev, S., and Strobel, S.A. (2001) Student Outcomes in a Local Systemic Change Project. School Science and Mathematics 101: 417-426.