PRINCIPLES OF BIOTECHNOLOGY (BIO-389/589)
SPRING 2006
PLACE AND TIME:
Halsey 367: 4:10 – 5:40 MW
INSTRUCTOR:
Dr. Toivo Kallas
OFFICE:
Halsey 245 (phone 424-7084; e-mail: kallas@uwosh.edu)
webpage (in progress): http://www.uwosh.edu/faculty_staff/kallas
OFFICE HOURS:
MW 11:30 – 12:30, 1:50 – 2:50. Other times by appointment.
Anytime by phone or e-mail. If I am not in, please leave a message
or check one of the lab rooms (HS 238, 240, or 163/145
Bioseparations-Proteomics Labs).
TEXTBOOKS AND RESOURCES:
Required:
1. Glick, B. R, Pasternak, J. J. 2003 Molecular Biotechnology. 3rd ed. ASM. Washington D.C.
(This text will serve mostly as a background and reference book.)
2. Much of the reading/discussion material for the course will come from journals such as
Nature Biotechnology, Trends in Biotechnology, Nature, Science, and others.
Recommended & Other Useful References:
(I strongly recommend a book on scientific writing such as #1 or #2 below):
1. Day, R. A. 1998. How to Write and Publish a Scientific Paper, 5th ed. Oryx Press, Phoenix.
2. McMillan, V. E. 2001. Writing Papers in the Biological Sciences, Bedford/St. Martin’s.
3. Primrose, S. B., Twyman, R. M. and Old, R. W. 2001. Principles of Gene Manipulation.
Blackwell, Oxford.
4. Crueger, W. and Crueger, A. 1989. Biotechnology: A Textbook of Industrial Microbiology.
Sinauer, Sunderland, MA.
5. Glazer, A. N. and Nikaido, H. 1995. Microbial Biotechnology. W. H. Freeman, New York.
(Some sections of Creuger and Creuger, Glazer and Nikaido, and other materials will be
available in a “reserve folder” in Halsey 240 or via electronic reserve.)
6. Sambrook, J and Russell, D. (2000) Molecular Cloning: A Laboratory Manual 3rd ed., Cold
Spring Harbor Laboratory.
Instructions for accessing the D2L (Desire2Learn) site: Go to the UW Oshkosh home page, >
then “Resources for: Current Students,” > then “Technology, Desire2Learn.” On the D2L login page, use
the username and password that you use for UW Oshkosh e-mail. Once in D2L, go to the Principles of
Biotechnology course to access lecture presentations, PDF files of articles, and other materials.
Some BIOTECHNOLOGY resources on the internet:
1. Class Web-site (in progress).
2. American Society for Microbiology (ASM) home page: http://www.asmusa.org
3. BioWeb http://bioweb.uwlax.edu/index.htm. (A nice collection of data and tools for genetics and
biology.
4. DOE Joint Genomics Institute, Microbial Genome Projects:
http://www.jgi.doe.gov/JGI_microbial/html/index.html
5. ExPASy Molecuar Biology Server: http://www.expasy.ch/. (A very useful site for molecular biology,
genomics, and proteomics included predicted peptide mass fingerprints.)
6. VIRTUAL GENOME CENTER: http://alces.med.umn.edu/VGC.html.
7. ***THE NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION: http://www.ncbi.nlm.nih.gov/.
(This site includes the GenBank and other DNA, protein, and genomic databases and extremely
useful search programs such as “BLAST.” Includes the PubMed, MEDLINE literature database.)
8. TIGR (The Institute for Genomic Research): http://www.tigr.org.
9. Kazusa Genome Research Institute: http://www.kazusa.or.jp. (Includes the database for the genome
sequence of the cyanobacterium Synechocystis PCC 6803.)
10. E. coli Genetics Stock Center: http://cgsc.biology.yale.edu/. (a useful site for gene names, maps, etc.)
11. E. coli Genome Center: http://www.genetics.wisc.edu:80/index.html
12. The Chlamydomonas Genetics Center: http://www.biology.duke.edu/chlamy/
13. The Human Genome Research Institute: http://www.genome.gov/
14. The RCSB Protein DATA Bank: http://www.rcsb.org/pdb/. (Site from which to download “.pdb” files
of coordinates for viewing and manipulating protein 3D structures).
15. WEBCUTTER (a nice site for on-line restriction site analysis): http://firstmarket.com/cutter/cut2.html.
16. NET PRIMER (a site that allows downloaded or on-line design of PCR primers. They also carry
“Plasmid Premier” a program for plasmid design): http://www.premierbiosoft.com/netprimer.html
17. SWISS-PROT, University of Geneva, Switzerland: http://expasy.hcuge.ch/sprot/sp-docu.html (Site
from which to download the Swiss-PDB viewer program for protein 3D structures).
18. RASMOL home page: http://www.umass.edu/microbiol/rasmol/ (Site from which to download the
RASMOL and PROTEIN EXPLORER programs for protein structure viewing & manipulation).
19. Frontdoor to PROTEIN EXPLORER: http://molvis.sdsc.edu/protexpl/frntdoor.htm (Site for online use
of the PROTEIN EXPLORER program for protein structure viewing & manipulation).
20. PyMOL: http://pymol.sourceforge.net/ (Site for downloading the PyMOL program for very nice viewing
and manipulation of protein and molecular 3D structures on Mac and Windows platforms.)
21. SINCRIS information server for crystallography: http://www.lcmp.jussieu.fr/sincris-top/ (A nice site for
information and access to programs and databases for viewing and manipulating biomolecules).
22. Nature Biotechnology: http://www.nature.com/nbt/, (available on-line via Polk Library)
23. Trends in Biotechnology: http://www.trends.com/tibtech/default.htm
24. New England Biolabs, Restriction Enzyme Database (NEB-REB): http://rebase.neb.com.
25. Promega Corporation (Madison, WI): http://www.promega.com/.
26. UW-O (Polk) Library: http://www.uwosh.edu/departments/llr/home.html. (Polk Library provides
access to a variety of useful literature databases such as Medline and Web of Science and carries online, full-text subscriptions to several relevant journals including Science, the Nature Journals
(including Nature and Nature Biotechnology, and the American Chemical Society (ACS) Journals.
Follow on-screen instructions. See the Polk Reference Desk or me for help.)
COURSE OBJECTIVES AND OVERVIEW:
Biotechnology represents the adaptation and uses of biological processes for practical (human)
ends. The roots of biotechnology date back to the dawn of civilization. Modern biotechnology draws upon
and has relevance to all fields of life sciences including animal, plant, cell, microbial, molecular biology,
biochemistry, genetics, and ecology. Central themes of the course include strategies for discovery of
novel biological products and production, recovery, and improvement of products and “bioprocesses”
through genetic and other means. Topics to be discussed include methods of screening for novel
bioproducts, principles of cell culture ("fermentation"), product recovery/purification, and enzymatic
"bioconversions." Special emphasis will be placed on concepts and applications of the "genetic
engineering revolution" which has allowed DNA manipulation in vitro and its reintroduction into living
organisms to yield genetically modified organisms and novel biological products and bioprocesses. We
will also discuss aspects of the ongoing revolutions in genomics, proteomics, and “bioinformatics,” which
are having tremendous impacts on life sciences and medicine.
Throughout the course, we will analyze and discuss selected research and review articles on current
topics in biotechnology. The objectives of this are: 1) to gain insight into methods and research at the
"frontiers" of biotechnology and 2) to learn about exciting current developments in diverse areas of
biotechnology such as protein engineering; gene expression in microbes and higher organisms; plant and
animal genetic engineering; DNA probe and polymerase chain reaction (PCR) technologies; microarray
and proteomic analysis of global gene expression and protein interaction networks, genetic diagnosis and
therapy; and environmental biotechnology.
GRADING AND REQUIREMENTS
Journal article reports
8 reports @ 10 points each (may submit 2
additional for extra credit)
2
80
Genome analysis/gene
expression exercise
due February 27
50
MIDTERM EXAM
March 3-10 (due March 10)
150
MINIREVIEW
due May 1
100
MINIREVIEW presentations
Week of May 1
50
Protein 3D structure exercise
due May 8
50
FINAL EXAM
May 5-12 (due May 12)
150
total
630
JOURNAL ARTICLE REPORTS: To encourage exploration of current topics in biotechnology, I am
requesting that each student find, read, and write brief reports (1 page or less) on current journal articles
related to biotechnology. You will need approximately one article per week (8 for the semester). These
reports should describe the objectives of the study reported in the article, the methods used, and the
main conclusions of the work. Further instructions will be provided. Some of these student articles may
be used subsequently for class discussion.
LITERATURE DISCUSSION–ANALYSIS: Usually one or two papers per week (from Nature
Biotechnology, Trends in Biotechnology, Nature, Science or other sources) will be assigned for class
discussion. You are expected to read these papers ahead of class and should be prepared to
summarize and discuss them in class. You will not be expected to, and may not, fully understand these
papers ahead of class, but you can improve your grade by participating actively and asking questions.
GRADING POLICY: 90-100% =A, 80-90% =B, 70-80% = C, 60-70% = D, less than 60%=F.
Grades of AB, BC, and CD will be used, at the discretion of the instructor, for borderline scores. If the
class scores on exams or assignments are uniformly low, grades may be adjusted accordingly. Exams
will consist of definition, problem, and discussion questions. Exams will typically be "open-book" and "takehome.” Undergraduates will be graded separately if graduate students consistently perform better on
exams and assignments.
MINIREVIEWS and PRESENTATIONS: Each student will write a minireview on a current topic in
biotechnology. Minireviews should be 5-10 manuscript pages long (~250 words/page and no longer than
10 pages) and contain 20 or more references (no more than 20% may be internet references). Details
and the format for the minireview will be described separately. To share minireview findings, each student
or pair of students will give a 15-20 minute presentation near the end of the semester. Graduate students
will give individual presentations.
LATE WORK: Late work will receive no more than 90% of full credit unless arrangements have been
made in advance.
ATTENDANCE POLICY:
Students are individually responsible for obtaining class materials,
completing exercises, and meeting course requirements. Advance notification of absences is expected.
CHEATING POLICY: We operate under the principle of "academic integrity" expected at this university. UW
System guidelines state: "Students are responsible for the honest completion and representation of their work, for
the appropriate citation of sources and for respect of others' academic endeavors." (s. UWS 14.01, Wis. Adm.
Code). Cheating or obstruction of the efforts of others will not be tolerated in any form. Students caught cheating
will receive an F grade and may be subject to further disciplinary action. Note in particular that this honor system
applies during take-home exams and assignments. Please do not be tempted to represent the work of others
as your own. This constitutes cheating (plagiarism) and will be treated as described above.
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TOPICS AND SCHEDULE:
(Glick and Pasternak (GP), ASM 2003, is the main reference text. Some sections of Primrose, Twyman, &
Old (PTO), Crueger and Crueger (CC), and Glazer and Nikaido (GN) are listed for reference. Relevant
materials from these are included in the Powerpoint presentations on D2L.)
(Other materials, e.g. literature discussion & review articles, will be available via the D2L site.)
Week
1
Topic
I. Introduction and course overview
Chapter
1-2 GP, 1-4 PTO
1-2
II. REVIEW OF BASIC GENETIC ENGINEERING TECHNIQUES
 Genetic basis and history of gene cloning
4-5 GP
(review 3 GP)
1 GP, 1-2 OP
1-2 GP, 1-2 PTO
4 GP, 3 PTO
4 GP, 4-6 PTO
 Restriction & modification of DNA, cutting & joining DNA molecules.
 Cloning vectors, host strains, DNA introduction into cells, selection
and screening for recombinants
 Polymerase chain reaction (PCR) & cloning applications
 Introduction: genome analysis – gene expression exercise:
NCBI site, Gene Construction Kit program, expression plasmids,
expression of cloned genes & protein products
 DNA sequencing & introduction to genomic databases &
bioinformatics
2-3
4-5
5-6
6-7 GP, 5, 9 PTO
5 GP, 7 PTO
III. BIODIVERSITY AND SCREENING FOR NOVEL BIOPRODUCTS
 Weird and unusual organisms and their biotechnological potential
 Molecular methods for exploring microbial diversity
 Genome sequences, genes, & bioproducts from “uncultivated”
organisms
3-4
5 GP
IV. Classical & molecular methods for screening & generation of
biodiversity
 Classical microbial & biochemical screening strategies
 "Smart screens" for discovery of novel bioproducts
 Mutagenesis strategies, directed random mutagenesis
 "In vitro" molecular evolution: “gene shuffling” & other methods
 Genetic tricks: bacteriophage and microbial surface display of
proteins
V. GENOMIC DATABASES, MICROARRAYS AND PROTEOMICS
 Genomic databases and microarrays for molecular diagnostics,
screening, and product discovery
 PCR, oligonucleotide, and “tiled” microarrays
 Genomic databases and proteomics for molecular diagnostics,
screening, and product discovery
 Identification of proteins & their modifications by: MALDI (matrixassisted-laser-desorption-ionization), ESI (electrospray-ionization),
and LC-ESI-MS/MS (liquid chromatography, electrospray tandem)
mass spectrometry
 Two-hybrid & protein array screens for probing molecular interactions
4
1, 2 GN
1-2 GN
parts of 8 GP
parts of 6, 10 GP
parts of 20 GP
1, 14 PTO
7-8
MIDTERM EXAM
March 3-10 (due March 10)
SPRING BREAK
March 11 – 19
VI. PRINCIPLES of CELL CULTURE FOR PRODUCT FORMATION
 Principles of "Fermenter" or bioreactor operation
(batch, fed-batch, and continuous cultures)




Biomass & ethanol, the potential of crude substrates
Introduction to microbial biotransformations & bioremediation
Metabolic pathway engineering
Examples of bioproducts and production strategies:
 Pharmaceuticals, enzymes, antibodies
 Vaccines
 Antibiotics, biopolymers
 Microbial insecticides
 Amino acids, vitamins, & small biological molecules
 Genetically engineered products
8-9
7 GP
10 GP, 7 GN
11 GP
12 GP, 8 GN
15 GP, 6 GN
12 GP, 13 GN,
6 CC
12 GP
VII. PRODUCT RECOVERY AND PURIFICATION
 Cell harvest, disruption, & primary separations
 Chromatography for separation of proteins & other biomolecules
 Ion exchange, “normal” phase, “reverse” phase, gel filtration, &
affinity chromatography
 High performance liquid chromatography (HPLC) strategies
 Biotech disasters & controversies, regulatory issues & genetically
modified organisms (GMOs)
 Advances in bioseparation strategies
 Fusion proteins & affinity purification tags
 Genetic engineering of protein conformation, stability, & export
9-10
16 GP
4, 5 CC
4,5 CC
13 GP, 10-11 GN
VIII. PROTEIN FOLDING, DEGRADATION, & MISFOLDING
(importance to biology, biotechnology, & medicine)
16 GP, 6 CC
21 GP
6-7 GP, 5, 9 PTO
parts of 6 GP
 Molecular chaperones, proteasomes, & foldases
 “Reporters” of protein folding & strategies for refolding misfolded
proteins
10-11
IX. ENZYMES & PROTEINS IN BIOTECHNOLOGY





Enzymes as bioproducts (e.g. in the molecular biology revolution)
Enzymes as biocatalysts
Enzymes in microbial transformations & bioremediation
Immobilized enzymes & enzyme biosensors
Protein 3D structures, databases, & structure viewing/manipulation
websites & programs
5
13 GP, 10-11 GN
parts of 9 GP
11-12
X. BIOSENSORS & MOLECULAR PROBES
 Organisms as biosensors
 DNA fingerprinting & probe techniques
 Restriction fragment length polymorphisms (RFLPs) & DNA
fingerprinting
 Allele-specific PCR
 “Molecular beacons” & Real-Time PCR
 Immuno-PCR
 Protein interaction probes: Fluorescence resonance energy
transfer (FRET)
 Nanobiotechnology: new approaches to molecular recognition
11-12
XI. FURTHER, ADVANCED GENETIC ENGINEERING STRATEGIES
 In vitro and site-directed mutagenesis
 RNA-based applications, e.g. interference RNA (RNAi)
 other selected, current topics
9, 20 GP
8 GP, 7 PTO
12-13
XII. TRANSGENIC PLANTS
 DNA introduction by “Agro-infection”
 Universal methods of DNA introduction: electroporation & particle
bombardment
 Genetically engineered foods & environmental concerns
 Applications of transgenic plants, examples
17-18 GP, 12PTO
12-13
XIII. TRANSGENIC ANIMALS
 Vectors & methods of DNA introduction
 Embryonic stem cells
 Somatic cells & reproductive cloning
 Applications of transgenic animals, examples
19 GP, 11 PTO
13-14
XIV. HUMAN GENE THERAPY, DIAGNOSIS, & MOLECULAR MEDICINE
9, 20-21 GP,
14 PTO




ex vivo & in vivo strategies
methods for transgene introduction & detection
role of genomics & proteomics
Embryonic stem cells, therapeutic cloning, & controversies
XV. ENVIRONMENTAL BIOTECHNOLOGY
13 GP, 14 PTO
XVI. ETHICAL AND PATENT ISSUES
21-22 GP
XVII. SELECTED CURRENT TOPICS (throughout the semester)
13
MINIREVIEWS
due May 1st
STUDENT PRESENTATIONS (week of May 1st)
TAKE-HOME FINAL EXAM
May 5 – 12 (due May 12)
End of semester celebration at Fratello’s! (May 12)
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