Is Teaching Biotech/Bioinformatics Worth It?

Is Teaching Biotech/Bioinformatics Worth It?

[

A Capstone Project

Presented to the Faculty of Science and Environmental Policy

in the

College of Science, Media Arts, and Technology

at

California State University, Monterey Bay

in Partial Fulfillment of the Requirements for the Degree of

Bachelor of Science

by

Bill Montgomery

April 4, 2006

Table of Contents

Abstract...... 2

Introduction...... 3

Methods

The 2005 MBB Workshop...... 4

Week 1...... 5

Week 2...... 6

Week 3...... 7

Analysis of the Activities Presented in the 2005 MBB Workshop...... 9

Results/Discussion of the Analysis of the Activities...... 11

Postcript: Interview with Susan Martimo...... 13

Bibliography...... 14

Appendices

A - 1990 Science Framework for California Public Schools...... 16

B - 2003 Science Framework, Biology/Life Sciences...... 17

C - 1998 Science Content Standards, Biology/Life Sciences...... 22

D - "Paper Primer" Activity...... 23

E - Modified DNeasy DNA Extraction Protocol...... 28

F - Agarose Gel Electrophoresis...... 30

G - Quantifying Sample DNA...... 32

H - Introduction to PCR...... 33

I - Species Identification of M. trossulus, M. galloprovencialis, and hybrids...... 35

J - PCR Troubleshooting...... 39

Abstract

To decide whether the challenge and expense of introducing Biotech/Bioinformatics curricula in secondary school is merited, a non-random sample of classroom activities is evaluated against existing state mandated content standards in California. Classroom activities presented in a teacher workshop for Marine Bioinformatics and Biotechnology were evaluated for their potential in addressing content standards of California Public Schools. The activities presented in the three-week workshop were structured around a research question concerning the status of an ongoing invasion by an introduced species of mussel (Mytilus). Even though almost 1/3 of the Life Sciences Content Standards for California Public Schools are on the subject of genetics, only 5 of those 22 content standards were considered to be addressed by the classroom activities presented in the workshop.Origins and relationship between content standards and frameworks are discussed.

Introduction

"I've got so much more to think about

Deadlines and commitments

What to leave in, what to leave out"

-Bob Seger, Against the Wind

Today's secondary school student really does have more to think about. When I was in high school in the early 80s, there were no computers there to learn about. Hubble hadn't been launched yet and we didn't know that there was a hole in the ozone layer. Just leafing through a 2003 Science Framework for California Public Schools, I see a lot of material that I wasn't introduced to until I was in college.

The relentless pace of scientific progress does not slow, it quickens. No other body of knowledge changes at the rate of the applied sciences. I took a graphic arts class in high school that has been made mostly obsolete by a $150 digital camera and a copy of PageMaker.

Each decade seems to bring the genesis of an entirely new science. This decade's most important breakthrough is, arguably, Biotechnology. Is it time to start teaching it in high school? Or do the legal requirements of our current educational standards prevent such an innovation?

In the 1990 edition of the Science Framework, the content standards for all grades (K-12) took 106 pages to print. In 2003, the content standards for grades 9-12 alone took 134 pages. I compare the content standards for the two editions in Appendices A & B. I omitted the 2003 edition's content standards for Mendelian Genetics, Meiosis, and Fertilization. None of these subjects were found in the 1990 edition.

With all this material to be learned, where would the time come from to learn a new technology such as Bioinformatics? Are the skills and concepts learned in the study of Biotech/Bioinformatics worth the extra effort?

I think that the first criteria for evaluating the "worth" of Biotech/Bioinformatics curricula is to judge it by the accepted teaching standards.

The 2003 Science Framework for California Public Schools makes several recommendations for matching instructional activities with content standards.

"In brief, teachers need to use instructional activities or readings that are grounded in science and that provide clear and nonsuperficial lessons. The content must be scientifically accurate and the breadth and depth of the science standards need to be addressed. Initial teaching sequences must communicate with students in the most straightforward way possible, and expanded teaching used to amplify the students' understanding. The concrete examples, investigative activities, and vocabulary used in instruction need to be unambiguous and chosen to demonstrate the wide range of variation on which scientific concepts can be generalized." (California Dept. of Education, 2003)

Therefore, in relating the activities demonstrated at the 2005 Marine Bioinformatics and Biotechnology Workshop, I will evaluate them according to the 2003 Science Framework for California Public Schools in regards to several criteria:

1. Does the activity address a content standard?

2. Is there content overlap with a similar activity(ies)? I have in mind to try to keep overlap to a minimum.

3. Does the activity demonstrate a concept that can be generalized?

The end result of this project will be to see how many of the 11 Molecular Biology and Biotechnology content standards were met by the activities that I encountered. I would expect to see all of the content standards met to decide that integrating Bioinformatics into any curricula that I would teach would be worth the extra effort (and expense) required. If I feel that an activity sufficiently addresses a content standard then I will consider criteria #1 above to have been met. The second and third criteria can then be evaluated to further evaluate the value of an activity in comparison to others.

If the activities from the workshop do not seem to meet all of the content standards then perhaps Bioinformatics is not an appropriate choice for secondary school science curriculum. Or perhaps a more systematic approach must be taken pedagogically to ensure that all of the science content standards are being met in such a way that the content of the science activities reinforce each other. This, I think, is what the recommendations made by the 2003 Science Framework are trying to ensure.

My background in Biotechnology began two years ago in second semester Biology. An assignment near the end of the semester required us to use resources at the website of the National Center for Biotechnology Information (NCBI, to find sequences for specific proteins in organisms of our choice and then compare the protein and gene sequences to similar organisms. I was astounded that there was so much data already in existence for a science that I was just being introduced to. And the data was free for the asking. It was a bit overwhelming but it was clear that Bioinformatics was a powerful set of tools.

The odyssey continued the following semester in Biochemistry. We used PDB Lite and Protein Explorer to find and view 3D models of Sperm Whale Myoglobin - another application of Bioinformatics.

In my final semester of classwork, I took the Biotechnology Lab course and learned to amplify DNA using PCR technology. I also learned how to design primers and run gels to separate DNA products for visual identification. The following summer is when I attended the workshop that I will describe.

The Workshop

The purpose of the Marine Biotechnology and Bioinformatics Workshop was to taketeachers through the hands-on process that scientists use in modern marine scientific researchfor the incorporation of marine Biotechnology and Bioinformatics concepts into their curricula. That it met it’s intended purpose is maybe best left up to the teachers who attended and the project’s Principle Evaluator, Dr. Helen Cagampang. What I want to look at is, “How much of the hands-on process is applicable to the state teaching content standards?”

1st Week

The 2005 Workshop ran from June 20 to July 8 for three weeks of 8-hour days. The first two days were spent on orientation, lab techniques and background lectures for a study on using Biotechnology to track the distribution of an invasive mussel (Mytilus sp.)

The scenario is that archaeological evidence indicates that the native mussel in protected embayments in California, Mytilus trossulus, was once found as far south as Santa Catalina Island. Today, M. trossulus is only found to the north of San Francisco Bay and has been replaced by the European invader, M. galloprovencialis. Since the morphology of the two species is so similar, a genetic assay is the best way to survey the success of the invasion on the coast near Moss Landing.

On unprotected rocks of the coast, another native, M. californianus is the dominant species in the area. We would collect mussels for out studies from a rocky shore area called “the jetty” and a protected tidewater area called “the bridge”. We would expect to find mostly M. californianus at the jetty and mostly M. galloprovencialis at the bridge.

On Day 3, we collected Mussels from the two disparate habitats that were only separated by about 500 meters. A dissection lab immediately followed with special attention paid to the sexual identification of the mussels. The reason for this is that Mytilus have an unusual transmission pattern of mitochondrial DNA. Females inherit mtDNA only from their mother, but they transmit it to both daughters and sons. Males inherit mtDNA from both parents, but they transmit to sons only the mtDNA that they inherited from their father. (Zouros et al, 1994). This inheritance is illustrated In the figure below. Although a male will have mitochondria from his mother, he won't pass it on to his offspring. The grey diagonal lines indicate that mitochondria from the parent above is present but not transmitted. The dark vertical lines show direct mitochondrial transmission from parent to offspring.

♂--♀ ♂--♀

| | /

♀--♂

| \ |

♀ ♂

As described, this pattern keeps the mitochondrial lineages separate although both lineages will be found in males (Skibinski et al, 1994). The presence of male mtDNA can thus be used to identify a male Mytilus. This will be important later.

The next day, we began an attempt to genetically identify the species of Mytilus that we collected. To do this, we amplified an Internal Transcribed Spacer (ITS) region of the rRNA gene in the DNA that we extracted from the mussels. The ITS loci have a high rate of divergence, even between species, are often used for species identification (James et al, 1996). Once the ITS regions have been amplified using PCR, they can be cut using a restriction enzyme that will create different sized products for each species. These products can then be run on a gel to show what size products are present.

Mytilus Genotype Scoring

Expected Results

Species / ITS

M. galloprovencialis

/ 200 & 450 bp

M. trossulus

/ 200 & 280 bp

Hybrid 1

/ 200, 280 & 450 bp

Unrestricted

/ ~950 bp

In the photo above, you can see how the EtBr tagged fragments sort themselves as they attempt to move vertically in the gel. The wells where the samples were placed are visible at the top. Each vertical lane is a separate PCR product sample. The distance moved vertically by each horizontal band corresponds to the ease with which that size fragment can move through the gel towards an oppositely charged pole. The number and size of the fragments observed in each lane identifies the species that the sample was taken from. I labeled one of each species and one of each of the three bp weights present. The two lanes on the right contain unrestricted DNA. The many-banded lane near the middle of the phone is a standard “ladder” sample used for calibration.

2nd Week

Week 2 began with an amplification of the gene for Cytochrome c Oxidase subunit 3 (CO3) prior to sequencing. This gene is commonly sequenced because it is mitochondrial and vital for respiration, thus also highly conserved and ubiquitous. COX, which catalyzes the transfer of electrons from reducedcytochrome c to molecular oxygen, is the primary determinantof cellular oxygen consumption and is thought to play a keyrole in regulating energy production (Poynton and McEwan 1996).

The protocol for PCR of the CO3 gene was very similar to the protocol for the ITS gene. Instead of analyzing the products directly this time, the PCR product of this protocol would be sent off for sequencing and the DNA sequences themselves would be analyzed. I won’t include the PCR activity for this because it really doesn’t add anything that hasn’t already been covered. The ITS protocol could be done on its own and if time or supplies didn’t permit a second protocol to be run, canned sequences could be used for the Bioinformatics part of the CO3 analysis.

We ran a couple of gels in the workshop to make sure that we had product before we sent it off for sequencing.

3rd Week

When our Mytilus CO3 sequences came back, we used Chromas software to save the output from the sequencer into a text string for the nucleotides in our samples. We cleaned up our sequences, edited them against their complements using Clustalx software, and then BLASTed them to see if they matched the sex and species of anything already in the database.

We submitted our resulting sequences to the PIs. We were shown how to produce phylogenetic trees in Clustalx and display them with DrawTree but we were also cautioned that trees made without research-level expertise were for reference only and not to be considered conclusive. That said, there’s something quite wonderful about watching these ancient relationships crystallize out of the sequence data.

Dr. Bartl chose the reference sequences that we would use to “ground” our mussel identification. These sequences were found in GenBank and trusted to be definitive for the species and sex of the source organism. We could then compare our new sequences to these standards. By drawing a phylogenetic tree of our new sequences and the reference sequences, we should be able to identify the sex and species of our new sequence by which reference sequence they grouped with.

Our reference sequences were:

Mytilus californianus: Accession # AF090831 (Beagley et al 1999)

Mytilus galloprovencialis (female): Accession # AF063264, species ID AG5F(Quesada et al 1998)

Mytilus galloprovencialis (male): Accession # AF063288, species ID AG4M (Quesada et al 1998)

Mytilus trossulus (female): The BLASTn of this sequence matched so many sequences in GenBank, that I couldn't tell which one was used. None of the species IDs matched the ones given on the tree.

Mytilus trossulus (male): Of the 11 sequence in GenBank that matched with no variations, 9 were for females and 2 weren't specified. Although a sequence was supplied to us for reference, it was not used to generate the tree.

The difficulty in finding a COIII sequence for M. trossulus was acknowledged by Dr. Bartl. The difficulty is compounded by the unique mitochondrial lineage of these organisms. It's possible to isolate a female mitochondrial gene in a male Mytilus. Dr Bartl suspects that's what happened in either the Genbank data or in our own efforts to identifiy a male M. trossulus. Thus the absence of a male M. trossulus in our phylogenetic tree.

So here is the phylogenetic tree from our 2005 workshop. I think that Dr. Bartl annotated it to clarify how our sequences were grouping with her "known" sequences from GenBank. The two M. galloprovencialis noted above (AG5F and AG4M) are near the center of their respective groups.

This tree doesn't show all of our sequences but none of the other trees that we produced showed any groupings with M. galloprovencialis males or M. trossulus. Even after the workshop, I continued to experiment with these procedures. The uniqueness of the mitochondrial lineage in Mytilus raises some interesting questions about why it might be so uncommon and what impact it might have on Mytilus adaptability.

So, what does this say about the status of our M. galloprovencialis invasion?

As far as Moss Landing is concerned, no M. Trossulus has been identified in the last two workshops. That's only 34 sequences, but our verdict for now is that the invasion is complete. The work at Moss Landing continues, though. I still scan the wire for new Mytilus COIII sequences. It was an exciting three weeks. I felt like I was a part of the research, not just a consumer. Not bad for a teacher workshop.

If nothing else, I learned what it takes to lab work exciting for students. It should be engaging. It should be a real question. Maybe even with an unknown answer. The closer you can get to what's really happening out there in the world, the more realistic the work seems.

The advantage of Bioinformatics in this case is that the lab resides in any computer that can surf to where the tools are. A lot of data is already out there. The questions are just waiting to be asked.

The Activities

.

Paper Primer Activity (Appendix D)

In this activity, students manually operate a pair of primer sequences that have been printed and cut out. They match up their primers to the places that they would bind on another printed sequence that acts as the sequence to be amplified. This is a very useful activity that teaches how DNA amplification is possible. In the Science Framework, there is no item that covers how DNA amplification technology works. Strand 5b covers naturally occurring semiconservative DNA replication.

Mytilus DNA Extraction(Appendix E)

The exact protocol that was followed is in the Appendix. The activity was prefaced by about a half-hour lecture on how this procedure extracts and amplifies DNA. This is a common procedure in Biotech for checking the quantity and quality of the DNA obtained and necessitates the skills practiced in the first lab activity above. As in the preceding case, this activity demonstrates a different technology than anything specified in the Science Framework.

Running a Gel (Appendices F & G)

Although strand 5d touches on gel electrophoresis, it does not do so in the context of a “check” of a successful DNA extraction. Nevertheless, this activity partially completes Life Sciences content standard 5d.

PCR and a Restriction Enzyme Digest (Appendices H, I & J)

In this activity we differentiated between two species of Mytilus by Polymerase Chain Reaction (PCR) amplification of a highly differentiated locus and then cutting that product with restriction enzymes to created different sized fragments that will tell us which species the DNA came from. PCR is another common and valuable skill to learn for Biotech. Getting DNA is one thing. Getting the exact piece of it that you want is another. This is good stuff. This activity combines accurate lab work with genetics, physics and chemistry. Activities that combine this many disparate sciences should really have a place in the Science Framework. Yet PCR is not even mentioned by the Science Framework. Using restriction enzymes to extract specific genes is, though. This activity also partially completes Life Sciences content standard 5d.