Unit 4 - Code of Life Part I: Genetic Engineering Biotechnology Part I: Genetic Engineering

Unit 4 - Code of Life Recombinant DNA Purpose: To insert genes of one organism into another organism

Recombinant DNA Applications Unit 4 - Code of Life Recombinant DNA Applications Green-glowing aquarium fish (jellyfish genes) http://news.bbc.co.uk/2/hi/science/nature/3026104.stm

Recombinant DNA Applications Fast-growing fish (Salmon with Pout genes) http://www.businessweek.com/magazine/content/06_03/b3967111.htm

Recombinant DNA Applications Herbicide-resistant crops (Round-Up Ready) http://www.pnas.org/content/103/35/13010.full Molecular basis for the herbicide resistance of Roundup Ready crops.Todd Funke�, Huijong Han�, Martha L. Healy-Fried�, Markus Fischer�, and Ernst Schbrunn�,ｧ+Author Affiliations.�Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045; and.�Department of Organic Chemistry and Biochemistry, Technical University Munich, D-85747 Garching, Germany.Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved July 12, 2006 (received for review May 3, 2006) Next SectionAbstractThe engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate has greatly improved agricultural efficiency worldwide. Glyphosate-based herbicides, such as Roundup, target the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely required for the survival of plants. Roundup Ready plants carry the gene coding for a glyphosate-insensitive form of this enzyme, obtained from Agrobacterium sp. strain CP4. Once incorporated into the plant genome, the gene product, CP4 EPSP synthase, confers crop resistance to glyphosate. Although widely used, the molecular basis for this glyphosate-resistance has remained obscure. We generated a synthetic gene coding for CP4 EPSP synthase and characterized the enzyme using kinetics and crystallography. The CP4 enzyme has unexpected kinetic and structural properties that render it unique among the known EPSP synthases. Glyphosate binds to the CP4 EPSP synthase in a condensed, noninhibitory conformation. Glyphosate sensitivity can be restored through a single-site mutation in the active site (Ala-100萌ly), allowing glyphosate to bind in its extended, inhibitory conformation.

Recombinant DNA Applications Unit 4 - Code of Life Recombinant DNA Applications Pest-resistant crops (Bt toxin)

Recombinant DNA Applications Unit 4 - Code of Life Medicine production (bacteria can make insulin, hGH, etc)

Making Recombinant Bacteria Important players: Gene of interest (i.e. insulin, hGH, etc) Restriction enzymes – cut the DNA Plasmid – circular DNA found in bacteria Bacteria

What are Restriction Enzymes? Target very specific base sequences Are found in more than 100 different varieties Are used in nature to protect bacteria from foreign invaders

What are Restriction Enzymes? Unit 4 - Code of Life What are Restriction Enzymes? Each restriction enzyme recognizes a very specific nucleotide sequence EcoR1 recognizes: GAATTC CTTAAG The enzyme cuts it: G AATTC CTTAA G

Unit 4 - Code of Life

Making Recombinant DNA Unit 4 - Code of Life Making Recombinant DNA Plasmids - self-replicating rings of DNA containing 2-30 genes, found in bacterial cells

Making Recombinant DNA Unit 4 - Code of Life Making Recombinant DNA Isolate the gene of interest using a restriction enzyme Cut the plasmid (using the same enzyme) Insert gene into the plasmid Insert the plasmid into bacteria Grow bacteria Harvest & purify the protein

Unit 4 - Code of Life 1. Isolating the gene Use restriction enzymes to cut the DNA strand at specific places that surround the gene

2. Cut the plasmid Cut the plasmid using the same restriction enzyme

3. Put the gene in the plasmid Since the gene and the plasmid have the same sticky ends, the gene will stick to the plasmid

Unit 4 - Code of Life The gene of interest is attached to a promoter and some kind of marker

5/6. Put plasmid in bacteria Insert plasmid into bacteria Let bacteria make the protein. Harvest protein & purifty it.

Unit 4 - Code of Life Part II: DNA Fingerprinting Biotechnology Part II: DNA Fingerprinting

Unit 4 - Code of Life DNA Fingerprinting Definition: Method of visualizing the differences in DNA samples Purpose: Forensic science Determining paternity Research Diagnosing Disease

Unit 4 - Code of Life Gel Electrophoresis Separates DNA fragments by size using electric current

Gel Electrophoresis Larger fragments move more slowly Unit 4 - Code of Life Gel Electrophoresis Larger fragments move more slowly Bands of fragments result

DNA Fingerprinting Method Unit 4 - Code of Life DNA Fingerprinting Method Use restriction enzymes to cut the DNA Unique DNA sequences produce unique banding patterns on the gel Load the DNA onto agarose gel for gel electrophoresis Analyze the banding pattern

Unit 4 - Code of Life

Gel Electrophoresis – Paternity Test

Gel Electrophoresis – Crime investigation

RFLP Analysis RFLP – Restriction Fragment Length Polymorphism: Restriction Enzymes used on two (or more) samples of DNA Different DNA fragments are produced because of variation in the DNA sequences Genetic markers are used for fingerprinting Gene sequences are used for genetic tests

A single nucleotide change can make a difference Wild-type allele AGATCT TCTAGA Restriction site Mutant allele AGAGCT TCTCGA Not a restriction site

Unit 4 - Code of Life http://children.webmd.com/video/sickle-cell-treatment – NOTE - WARN STUDENTS OF THE END WHERE THE AFRICAN-AMERICAN BOY IS MAKING FRIED CHICKEN (MY AASP STUDENTS THOUGHT THIS WAS RACIST) http://children.webmd.com/video/sickle-cell-cure