Biotechnology: Methods

Biology 100/101
Lecture 18:
Biotechnology: Methods

Text Readings
in Lewis

p. 334-335; 453-454

Text Readings in Lewis
p. 334-335; 453-454

The text book has limited information concerning the topic of biotechnology so we have tried to provide web links that should provide the needed background information. I hope you find them to be interesting and understandable. We will supplement this information with activities in lab and discussion to help you understand biotechnology.

For feedback, post possible answers and ideas in the folder "Text 'Review' and 'To Think About' Questions" in the Biology Chat Section of Web Crossing.

Objectives
After studying this material you should be able to:

Describe the steps leading to a recombinant protein, including;
1. ) constructing a gene library
2. ) identifying a gene in a gene library
3. ) cutting outthe gene and ligating it into an expression vector, and
4. ) then transforming the expression factor (plasmid) back into bacteria.
Explain why restriction enzymes are such useful tools in biotechnology.
Link the following issues in a concept map when describing how recombinant insulin is made in bacteria, OR how one might genetically modify a human to rectify a genetic disease that results form a protein deficiency (i.e. "repairing" a mutation that blocks the expression of a needed protein, by adding back the wild-type (normal) gene).
1. ) identifying a gene of interest,
2. ) placing that gene in a plasmid that allows you to amplify the gene and to maintain it in bacteria,
3. ) constructing a vector that allows you expression the gene product in a desired organism (can be with an expression plasmid or by inserting the "gene construct" directly into the organism's genome),
4. ) using an appropriate promoter.

Key Terms

recombinant DNA
restriction enzyme (endonuclease)
palindromes
ligase
plasmid
expression vector
transformation
transgenic technology
nucleic acid hybridization
recombinant protein

General Web resources:

Understanding Gene Testing
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service National Institutes of Health National Cancer Institute
Accessed from Access Excellence a web site managed by Genentech, a biotechnology company in California
These sites contain a wealth of information concerning genetics, DNA, and Biotechnology. I will include links to specific topics in the body of the outlines for this lecture and lectures 18 and 19, however I invite you to explore on your own. There might be potential net surfer projects here.
Access Excellence Graphics Gallery
Principles Of Biotechnology North Central Regional Extension Publication Iowa State University - University Extension
Biotechnology - The Future from Access Excellence
The Washington Biotechnology Action Council This organization provides an alternative view of the potential benefits of biotechnology
Enter a keyword or words to search (Access Excellence) A Wealth of Biotechnology Links

Recombinant DNA Technology:

Altering the genetic makeup of an organism by:

adding new DNA to it, or,

changing the DNA that is already there.

What are the tools of Biotechnology?
RESTRICTION ENDONUCLEASE "SLIDE"
DNA that has been cut with a restriction enzyme can be put back together.
DNA LIGASE REPAIRS BREAKS IN DNA "SLIDE"
The power of recombinant DNA technology is that a piece of DNA cut from a cell's genome can be joined with a new section of DNA.
!!!Restriction Enzyme Action (Access Excellence)!!!
What is this new section of DNA and why is this useful??
- Plasmids are self-replicating, circular DNAs found in many organisms. They are NOT part of that organism's chromosomal DNA, but a sort of "free-loader" in the cell. These plasmids can be used as tools for expressing foreign genes in transformed organisms.
  (Plasmids are also why many antibiotics are in danger of losing their effectiveness because many plasmids contain genes for antibiotic resistance)
  PLASMIDS & PHAGE "SLIDE"
HOW can plasmids be used as tools for expressing foreign genes in transformed organisms?
Easy, paste foreign DNA into the plasmid and then get that plasmid back into a cell.
CUTTING AND PASTING DNA WITH ENZYMES "SLIDE"
DNA and plasmids are cut and ligated together in a test tube.
How do we get them back into a cell?
With transformation, using chemicals, electric shock, or even "gene guns" to transfer plasmids into recipient cells.
GENETIC TRANSFORMATION "SLIDE"
Some specific genetic transformation techniques and examples are presented in this series of links:
Once a cell is transformed with a desired gene, it can be induced to express that foreign protein.
This is possible because the genetic code and basic machinery for protein biosynthesis are universal.
One simply puts the desired gene behind an appropriate promoter (remember a promoter contains the information that controls the "expression" of a gene) for that organism.
EXAMPLE: THE PRODUCTION OF INSULIN

DNA
(Human
Insulin Allele)
in Bacterial Plasmid --> Transcription
(in bacterium) --> Insulin
mRNA --> Translation
(in bacterium) --> Protein
(Insulin)
harvested
from bacteria
!!!Transfer and Cloning of the Insulin Gene (Access Excellence)!!!
Additional text and web resources to help you understand restriction enzymes, plasmids, and recombinant DNA:

Identifying Specific Genes

Constructing a DNA Library: A tool for identifying specific genes.
How are these DNAs used:
1. ) to learn about the encoded protein
2. ) to produce recombinant protein for bio-medical needs
3. ) to "engineer" an organism for an improved trait or repair a faulty one
  DNA LIBRARY "SLIDE"
4. The DNA library contains at least one copy of every gene in that organism.
  How do you identify the gene of interest in these millions of transformed bacteria??
5. Nucleic Acid Hybridization: Forming a DNA-RNA hybrid.
6. Millions of bacteria with recombinant plasmids can be screened with small "man-made" RNA molecules produced in a test tube, whose sequence was deduced from protein sequence and the genetic code.
  In addition, the RNA has a label so we can find it after it hybridizes with DNA. Thus, a full length copy of the original gene that codes for a specific protein is obtained.

Additional text and Web resources for biotechnology
(Review of this lecture and a preview of the next one):

Restrictriction Enzymes, RFLPs, Electrophoresis, and Probes
- !!!Web Crossing Supplement - Electrophoresis and DNA Analysis!!!
- How do scientists develop predictive gene tests? (Access Excellence)
- Restriction Enzyme Cutting DNA (Access Excellence)
- Autoradiograph of electrophoresis gel from The National Human Genome Research Institute
- See Lewis' text, pages 314-315.
  - DNA Probes
  - RFLPs (Restriction Fragment Length Polymorphisms)
  - Electrophoresis
Transgenic Organisms
- Is Making Clones Making Trouble, (from the Exploratorium)
- Dolly: An Unsettling Breakthrough (from Pathfinder.com - TIME on-line)
- Green Genes Gerald R. Fink, Ph.D. Looking for ways to produce stronger, healthier, more marketable plants with DNA technology (From Access excellence)
  This is a series of web pages that looks at ways of incorporating new genes into plants.
- The Biopharmaceuticals -FDA-Approved Biopharmaceutical Drugs and Vaccines
- Herman the Bull
- Enter a keyword or words to search (Access Excellence)
  Do a search here for "transgenic" for a definition and more information about transgenic organisms.
Take me home.