Facilitating the Integration of Biotechnology Experiences into Diverse Undergraduate Courses

Peter Jankay, Cal Poly, San Luis Obispo, CA pjankay@calpoly.edu

3. A. Readily available equipment – Mobile Laboratory Common to most protocols from diverse biotech applications are three

distinct steps: DNA isolation, PCR, and gel electrophoresis.

All of the equipment for a given step (equipment module) can be loaded onto a cart, and thus can be wheeled literally anywhere on campus.

As an example, the electrophoresis mobile module contains everything for making gels (microwave, balance, graduated cylinders, buffer, etc), six gel boxes, power supplies, pipetors, tips, ethidium bromide, and a gel documentation system for a class of 24 students. See figure lower left.

So how is the mobile laboratory concept any better than a traditional molecular laboratory? The traditional laboratory has all necessary equipment located in a single room, e.g., a molecular laboratory. When being used by one class, none of the equipment is available to any other class. In contrast, the mobile laboratory can simultaneously serve different classes at different locations. The mobile laboratory, therefore, makes equipment readily available in a highly efficient manner.

The user need not even have a sink. See figure lower left of middle  partially showing a classroom set up for freshmen.

2. Training. UBL trains faculty and graduate student TAs how to teach course specific exercises. Trainees learn the techniques, are required to perform each step, are given basic theory for each step, are given proven logistics for the classroom, are given lessons learned by others who have taught the course. Last year alone we trained four faculty, and 21 graduate student TA’s.

The UBL Technicians

Two undergraduate students are hired each year who can each make a ten-hour/week commitment from September through June, and who have had a course like Molecular Biology Laboratory. These undergraduate students play very important roles for UBL. They

develop, and trouble shoot course specific exercises

quality test solutions to be used by various courses

train faculty and graduate student associates

train and assist undergraduates in the pursuit of their research projects that involve biotech applications.

operate the ABI 377 DNA sequencer for students working on independent projects and for classes who have completed the cycle sequencing. Especially for classes, the technicians give instruction on loading the gel comb, the technicians “drop the loaded gel comb in,” and using existing gel files they give abbreviated theory and a tour of the operation the sequencer.

operate the real time PCR machine for classes, and for students working on independent projects.

gel boxes

gel doc system

pneumatic wheels

power supplies

1 2 3 4

5 6 7 8

9

microwave

goggles

spatulas

flasks

tongs

pipetors balance

buffergloves

tips

agarose

IntroductionBiotechnology is pervasive in applied and basic science and its impact on society is great. The undergraduate biotechnology laboratory (UBL) was created to facilitate the appropriate integration of biotechnology into the undergraduate curriculum.

Cal Poly undergraduate programs prior to UBL (1999)

I. Very few undergraduate research projects on campus could address questions that needed basic PCR, DNA sequencing, or blot sensitivity.

II. The biological sciences department’s molecular bio lab course essentially ignored sequencing and bioinformatics. Our vertebrate development lab only looked at prepared slides and models.

III. Our intro to bio majors course did not address concepts like PCR. None of the 2K plus non-major students annually were introduced to concepts like RNA processing.

IV. No student in the Crops Science, Animal Science, and Physics departments encountered any biotechnology in any class or research project, and none of these faculty had access to any biotech tools.

Cal Poly undergraduate programs with UBLI. Regardless of their faculty advisors’ time availability, expertise,

or equipment, students now address questions that require tools like PCR and DNA sequencing, e.g.,

II. The molecular bio lab course now sequences plasmid inserts, and uses Lasergene software, and NCBI databases. UBL is in the beginning phase to help develop an exercise for the vertebrate development course that uses qPCR to address patterns of gene expression.

III. About 1K freshman science majors and about 2K freshman non-science students annually each isolate DNA from their own cheek cells, and perform PCR and electrophoresis to determine their own pv92 genotype. As part of the exercise, students address questions regarding PCR technology, gene structure and regulation, RNA processing, and the significance of transposable elements.

IV. The Animal Sciences department developed an animal biotechnology course. The Crops department developed lab exercises for their intro students that use PCR to answer real questions important to the discipline, e.g. “Did Dr. Phillips illegally plant Round-up Ready cotton?” Both departments hired faculty with considerable biotech expertise.

Created a gene construct to be used for phytoremediation of oil contaminated soil.

Determined if he inherited his mother's breast cancer gene.

Tetrahedral Photonic Bandgap Crystal: Three-Dimensional Self-Assembly using DNA Linkage

UBL was successful. What obstacles did UBL have to overcome?

Teaching loads are heavy, and research expectations are increasing.

Limited budgets since 1985 left the department with little biotech equipment.

Faculty (new and "seasoned") were quite reluctant to incorporate biotechnology into their classes even if they had the expertise, and even if the department were to acquire equipment. Probable reasons include perceptions like:

equipment would not be readily available for their course

it would consume considerable time to develop an appropriate exercise

experiments would have a high failure rate

it would take too much to bring themselves up to speed with the application

experiments would be at the cost of eliminating traditional exercises in the course

1. considerable assistance to develop exercises, encouragement, and assurance that the exercises will work in the classroom without extraordinary efforts on their part and without losing too much of the previous course content

2. training3. readily available equipment

1. Course specific exercises. UBL had to be proactive. First we had to identify courses whose topics result from basic or applied research making use of biotech tools.

Individual faculty were contacted and encouraged to work with UBL to develop objectives that focus on questions/issues important to their course

UBL then adapts and rigorously tests protocols for higher probability of success in classroom. This includes anticipating common mistakes, and misuses by students who often have limited or no biotech experience.

UBL troubleshoots problems when they arise

Examples of exercises are:⇝Freshman majors and non-majors bio courses: Alu polymorphism (see figure to the right)

⇝Fisheries: Identification of rockfish species. (see below right)

⇝Plant biotechnology: Gene discovery using activation tagging.

⇝Weed Science: What is the herbicide resistance mechanism of rye grass?

How does UBL facilitate undergraduate research? In an outreach approach, UBL’s director talks with faculty about their research interests and about interests and ideas their students have had. This led to undergraduate projects not previously even considered. Awareness about UBL’s services are also spread by word of mouth.

UBL director discusses the project with the student's advisor.

Students make appointments with the UBL technicians for equipment use and assistance.

UBL technicians train students in use of the equipment and protocols.

Student needs vary, e.g.,•Students working on the malaria parasite in lizard populations had absolutely no skills.

•Their faculty advisor is trained but simply did not have time.

•UBL technicians trained the students with how to: obtain blood samples, use the DNA isolation kit, determine DNA quality and quantity, calculate primer dilutions, perform PCR, electrophoresis, print and digitally save gel for later annotation.

•After training, students are permitted to work independently in the stationary lab. However, they are free to ask for additional assistance whenever they get stuck.

•Sometimes students need help only in trouble shooting a protocol or help with optimization, e.g., PCR conditions.

3. B. Stationary Laboratory This laboratory houses a complete set of equipment that duplicates mobile equipment (e.g., thermocycler, gel boxes, gel documentation system, balance), as well as non-mobile equipment like the ABI 377 DNA sequencer, and BioRad’s Fluorimager. The stationary laboratory supports, development of course specific exercises, training, and undergraduate research activities.

How did UBL overcome the obstacles and get faculty involved? It was obvious that whether or not faculty had recent molecular experiences, faculty needed:

Initial Funding. This project was started with funding from a Cal Poly grant, an NSFCCLI grant, and a CSUPERB grant.

Continued funding Salaries. Cal Poly colleges, Science and Mathematics, and Agriculture jointly fund the two undergraduate student technician positions. The director works receives an occasional pat on the back.

Expendables. Costs of expendables for course exercises can be rather significant. It is estimated that it costs about $6 per student to perform the Alu polymorphism exercise; note that approximately 2,000 students perform this exercise annually.

The enlightened Administration has approved a three-tiered course fee system for courses that use one or more biotechnology application.

Applications of DNA Technology in Marine Fisheries Biology

The marine resources of California include a large group referred to as “Rockfishes” that belong to the Scorpionfish family.

Canary rockfish (Sebastes pinniger )

Quillback rockfish (S. Maliger )

Black rockfish (S. melanops )

Redbanded rockfish (S. babcocki )

There are over 59 species along the California coast, many of which are economically important, both commercially and recreationally.

Some of these species have undergone precipitous declines in recent years.

In solving these problems, marine biologists need to be able to estimate population size and make projections of long term trends.A traditional way of doing this is to systematically sample the ocean for eggs and newly hatched young with devices such as plankton nets.

The life history of rockfishes is very complex and includes early planktonic stages that are difficult to identify to species.

Modern molecular biology has provided new ways to distinguish the planktonic stages of the various rockfish species.The following graphics demonstrate the application of RFLP (Restriction Fragment Length Polymorphism) analysis of three species of rockfish. The work was performed in the Fisheries Science and Conservation course (BIO 423) during the winter quarters of 2000 and 2001.

Fin tissue samples were taken and analyzed by gel electrophoresis to distinguish variations in the fish DNA.

1) Genomic DNA was isolated from fin tissue.2) The sequence was amplified using PCR (Polymerase Chain Reaction).3) PCR products were digested with an endonuclease4) The digested PCR was resolved using gel electrophoresis.

The results show clear separation of the three species on the basis of gel banding patterns.

Can you see the three distinct banding patterns for the three species?

With this technology a marine biologist can now make a positive identification of rockfish larvae sampled of the California coast.Continuing work on gene sequencing and microsatellite analysis on these species could provide additional information on important and often controversial questions such as:

Do populations associated with coastal marine preserves contribute significantly to the conservation of fished populations?

Does an individual rockfish come from a large, single, genetically homogenous, population distributed along the Pacific coast?

The curriculum in the Marine Biology and Fisheries Concentration exposes students to both theoretical and applied aspects of marine science, including current issues and controversies.

Olive rockfish (S. serranoides)

Yellowtail rockfish (S. flavidus)

Blue rockfish (S. mystinus)

Poster: Todd Olive Royden Nakamura Peter Jankay

Acknowledgements: National Science FoundationCal Poly PlanSouthwest Fisheries Science Center (National Marine Fisheries Service / NOAA)

Winter 2002 Alu Polymorphism

BIO 115 T3-6

BIO 111 T8-11 BIO 111 T12-3 BIO 111 T3-6 BIO 111 T7-10 BIO 111 W8-11

BIO111 R12-3BIO 111 R8-11BIO 111 W7-10BIO 111 W3-6BIO 111 W12-3

BIO111 R3-6 BIO 111 W 8-11 BIO 111 F8-11 BIO 111 F12-3 BIO 151 T/R12-3

BIO 151 T/R7-10BIO 151 T/R3-6 BIO 115 T8-11

BIO 115 W8-11 BIO 115 W12-3BIO 115 T12-3

BIO 115 R8-11 BIO 115 R12-3

BIO 151 W/F12-3

BIO 115 R3-6

BIO 151 T/R8-11

BIO 115 R7-10

BIO 115 W3-6

Each panel shows all the individual students in one lab section.

First, students removed some of their cheek cells and isolated the DNA from these cells.

Second, students used PCR to make millions of copies of a regionof chromosome 16 that may or may not have had a “jumping gene.”

Third, students used gel electrophoresis to separate DNA according to size. Pieces of DNA without a “jumping gene” would have been 550 bases long. Piecesof DNA containing the “jumping gene” would have been 850 bases long.

Here are the following genotypes:

Person homozygous with the “jumping gene”

Person homozygous without the “jumping gene”

Person who is heterozygous --

(one chromosome 16 has the “jumping gene,”

the other chromosome 16 does not)

www.bio.calpoly.edu/ubl