National Science Foundation Instrumentation and Laboratory Instrument Support

- Please feel free to use this as a model for your grant proposals.

The following report explains how the Ventura College Biotechnology Program has been able to utilize National Science Foundation Instrumentation and Laboratory Instrument funding. The $20,000 award required an equal match of funds, which are documented in the final report. You are welcome to use this report as a model. An abstract of the grant is available at www.nsf.gov, and a full copy can be obtained from the author (Professor Bill Thieman) by a request through email or telephone. btheiman@ventura.cc.ca.us or (805) 648-8954.

Major revisions were made in the lab experiences of students enrolling in biology labs and for the biotechnology/molecular biology training program. The NSF funding provided students with a comprehensive biotechnology lab program and improved instrumentation in biology labs. The lab exercises gave students hand-on experience in lab problem solving activities.

The first offering of the Introduction to Biotechnology and Molecular Biology Instrumentation course (Biology 31) occurred in the Spring of 1997, with 16 student graduates. Tracking of these students and of the second section (offered Summer 1997) resulted in a 42% rate of hiring into (mostly) entry-level biotechnology positions. This measure of success is low since most of the other students decided to continue their education and were not counted as "placed" by our tracking system.

The addition of this course (Biology 31) to the biology program made it possible to award 6 Certificates of Completion to students who satisfactorily finished all the courses in the program. Eleven new lab exercises were added to biology courses, providing a learning environment significantly enriched in molecular biological methods.

BIOLOGY 31 EXERCISES THAT UTILIZE NSF INSTRUMENTATION

1. LAB SKILLS: This is a typical lab orientation activity including safety and solution preparations. The NSF/grant-purchased analytic balance has made it possible for students to obtain two decimal place accuracy in the preparation of molar and normal solutions that verify their buffer and solution calculations. This skill is essential to employment in the biotechnology industry, and in preparation for lab experimentation.
   
   
2. MICROBIOLOGICAL METHODS: Each student is expected to perform a gram stain and evaluate the results. They are also expected to perform a serial dilution and determine the accuracy based on spectrophotometric evaluation of a dye present in the standard. The NSF/grant- purchased micropipetters and DUV21 spectrophotometers improved the measurement accuracy of the serial dilutions.
   
   
3. PROTEIN DIVERSITY: Each student performed an SDS PAGE electrophoresis exercise to determine the molecular weight of unknown proteins by comparison to standards. They learn to accurately pipette the protein samples and run the electrophoresis gel. The NSF purchased micropipetters and the Novex miniprotein II gel boxes and power supplies have made this commonly used method for protein characterization a successful learning tool. The Novex 12% pre-poured polyacrylamide gels were donated by a local biotech company (beyond expiration date). Using pre-poured gels reduces the danger of handling acrylamide for students, and reflects common practice in the biotechnology industry. In addition, protein standards are used to produce a standard curve of concentrations, analyzed using the Bradford assay method. The NSF-purchased DUV 21 Spectrophotometers and micropipetters are used in this analysis, as each student must determine the expected protein concentration and measure these from the proper dilutions of the assay.
   
   
4. EXTRACELLULAR PROTEIN EXPRESSION: This exercise has been adapted from a kit supplied by a lab supplier. It requires an overnight culture of Pseudomonas that can be induced to express the enzyme agarase. Students pour their own agarose plates and innoculate these radial diffusion plates cultures induced in the presence of agarose with both lysed bacterial pellet and supernatants of these cultures. Measuring the zones of turbidity (from digestion of the agarose) indicates that induced bacterial form supernatants that have the highest concentration of the expressed enzyme. The NSF/grant-purchased laminar flow hood and vortex shakers made this exercise possible.
   
   
5. RADIAL IMMUNODIFFUSION: This exercise involves the preparation of agarose plates with the addition of a known antibody. The antigens and control are added to wells cut by the students into the solidified agar and are of increasing antigen concentration. The antigen diffuses into the agar until its concentration matches that of antibody, forming a precipitation ring. Students calculate the distance of migration and plot this distance on a graph that relates distance to concentration of antibody. The exercise is very reliable. The NSF/grant-purchased micropipetters make this exercise a successful learning experience for students.
   
   
6. IMMUNOELECTROPHORESIS: This exercise involves the electrophoretic migration of serum antigens in a horizontal agarose gel, followed by the diffusion of antibodies to these antigens. A large number of arcs of precipitation occur between anti-serum antibodies and whole serum compared to the single antigen/antibody reaction for IgG and anti-IgG . The student results indicate the effect of concentration and the presence of specific binding. The NSF/grant-purchased horizontal gel boxes, power supplies and micropipetters made this a successful learning experience for students.
   
   
7. ANIMAL TISSUE CULTURE: This exercise involves the use of the cryo-freezer (tissue storage), laminar flow hood and the inverted microscope, that were NSF/grant purchased. Students aliquot quantities of HeLa cells from frozen culture and determine the dilution needed to innoculate 1 million cells into 10 ml of growth culture media. Hemocytometers are used and trypan blue is added to determine the number of viable cells. The exercise is on-going, since the cultures must be sub-cultured when they reach confluence (determined by observation with the inverted microscope). The skills of aseptic transfer in a laminar hood are valuable for students that are interested in positions in the biotechnology industry.
   
   
8. THIN LAYER CHROMATOGRAPHY: This exercise involves the separation of plant pigments on adsorption plates. The extraction is performed by the students and the plates are run in benzene/ether in micro-quantity. The separation is based on solvent adsorption to the plate, solubility, and the negative effects of gravity. The ratios of distances of migration are analyzed. The NSF/grant-supported micropipetters are needed in this exercise.
   
   
9. PLASMID FUSION: This exercise involves the fusion of two plasmids from pUC18 and pBR325 each containing a different antibiotic resistance gene (AMP and KAN). The restriction enzymes are added to the reaction tubes, followed by DNA ligase and transforms are produced. Competent E coli are induced to take up the transformed plasmid in the presence of calcium chloride and a heat shock. The plated cells are selected by their marker ability to grow on the culture media containing both antibiotics. The NSF/grant-purchased micropippeters and the laminar flow hoods are invaluable to the success of the exercise.
   
   
10. PLASMID MAPPING: This exercise results from students cutting pUC18 and pBR325 with restriction enzymes AatII and Sph 1. The students must predict the results of the exercise based on the known size of these plasmids and the number of cut sites. The cut plasmids are separated on agarose gels and the fragments are visualized after ethidium bromide staining. The results of the digestion are analyzed after each student photographs their gel with the NSF/grant-purchased horizontal gel boxes and UV illumination box and camera (the Epindorf microfuge is another necessary piece of equipment for this exercise that was grant purchased). The success of the exercise comes from the unexpected uncut and double ring plasmids that usually show up as bands on some student's gel.

    The Majors Biology class (Biology 20) utilized the plasmid fusion and plasmid mapping exercises described above. They also used the high speed centrifuge to perform an isolation and assay of mitochondria.

11. ISOLATION OF MITOCHONDRIA FROM CAULIFLOWER: Cauliflower rossettes are cut and ground in a mortar/pestle. The nuclei and cell debris is pelleted by centrifugation followed by high speed centrifugation of the supernatant (containing the mitochondria). The mitochondria are re-suspended in assay buffer and verified using phase microscopy. The non-mitochondrial supernatant and the mitochondrial pellet are assayed for succinate dehydrogenase activity using a technique from a lab manual by Holley Ahern (Molecular Biology). The transfer of electrons to the dye occurs because the final electron acceptor (cytchrome oxidase) is blocked by the presence of azide. The NSF-grant supported purchase of the Epindorf microfuge and micropipetters make this exercise a successful addition to the Majors Biology course.

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