1. Ms. Gaynor Honors Genetics Biotechnology and the Use of Bacteria

2. What is Biotechnology? The use of microorganisms to create cellular products (ie: insulin) for human use The use of microorganisms to create cellular products (ie: insulin) for human use Also known as genetic engineering Also known as genetic engineering Often uses bacteria as the microorganism Often uses bacteria as the microorganism

3. What is Bacteria? Single celled prokaryote Single celled prokaryote No nucleus (nucleoid region instead) No nucleus (nucleoid region instead) Very few (“no”) organelles Very few (“no”) organelles Has a cell wall, cell membrane, ribosomes, cytoplasm Has a cell wall, cell membrane, ribosomes, cytoplasm Has circular chromosome and plasmid(s) Has circular chromosome and plasmid(s) http://media.pearsoncmg.com/bc/bc_campbell_biology_7/media/inter activemedia/activities/load.html?6&B http://media.pearsoncmg.com/bc/bc_campbell_biology_7/media/inter activemedia/activities/load.html?6&B http://media.pearsoncmg.com/bc/bc_campbell_biology_7/media/inter activemedia/activities/load.html?6&B http://media.pearsoncmg.com/bc/bc_campbell_biology_7/media/inter activemedia/activities/load.html?6&B

4. 2 Types (domains) of Bacteria Eubacteria (can be harmful) ***used in biotechnology! Eubacteria (can be harmful) ***used in biotechnology! “regular” bacteria “regular” bacteria They live in most environments They live in most environments their cell-wall DOES contain peptidoglycan their cell-wall DOES contain peptidoglycan Archaea Bacteria (not harmful) Archaea Bacteria (not harmful) “older” bacteria; live in EXTREME habitats “older” bacteria; live in EXTREME habitats more similar to eukaryotes than to bacteria in several ways: more similar to eukaryotes than to bacteria in several ways: their cell-wall does NOT contain peptidoglycan their cell-wall does NOT contain peptidoglycan

5. Plasmids are used in Biotechnology In addition to the chromosome, some bacteria have plasmids In addition to the chromosome, some bacteria have plasmids – smaller circular DNA molecules Extra chromosomal DNA Extra chromosomal DNA

6. How Does Bacteria Get Plasmid DNA? Transformation Transformation Bacteria takes in naked, foreign DNA ( a plasmid) from the environment under certain conditions Bacteria takes in naked, foreign DNA ( a plasmid) from the environment under certain conditions http://www.phschool.com/science/biology_place/labbench/lab6/concepts1.html http://www.phschool.com/science/biology_place/labbench/lab6/concepts1.html http://www.phschool.com/science/biology_place/labbench/lab6/concepts1.html

7. DNA Technology DNA technology has launched a revolution in the area of biotechnology DNA technology has launched a revolution in the area of biotechnology The manipulation of organisms or their genetic components to make useful products The manipulation of organisms or their genetic components to make useful products Example: gene cloning Example: gene cloning

8. What is Gene Cloning? DNA cloning permits production of multiple copies of a specific gene or other DNA segment DNA cloning permits production of multiple copies of a specific gene or other DNA segment To work directly with specific genes To work directly with specific genes Scientists developed methods for making pieces of DNA in multiple identical copies, a process called gene cloning Scientists developed methods for making pieces of DNA in multiple identical copies, a process called gene cloning Usually uses bacterial plasmids Usually uses bacterial plasmids

11. Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes Bacterial restriction enzymes Called restriction endonucleases Called restriction endonucleases Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites

12. Restriction enzymes Where are restriction enzymes found? Where are restriction enzymes found? In bacterial cells, to help fight viruses/ foreign DNA In bacterial cells, to help fight viruses/ foreign DNA What is a restriction site? What is a restriction site? The area on DNA that the restriction enzyme recognizes The area on DNA that the restriction enzyme recognizes Every time this DNA sequence occurs in bacterium’s own DNA, methyl groups (-CH3) are added  prevents restriction enzyme from working. Every time this DNA sequence occurs in bacterium’s own DNA, methyl groups (-CH3) are added  prevents restriction enzyme from working. When foreign DNA (ex: a bacterial virus) enters bacterium, bacterium’s restriction enzyme cut phage’s DNA into pieces. When foreign DNA (ex: a bacterial virus) enters bacterium, bacterium’s restriction enzyme cut phage’s DNA into pieces. NOT all bacteria have restriction enzymes, many do! NOT all bacteria have restriction enzymes, many do! But there is a wide variety of restriction enzymes that have been discovered and continue to be discovered But there is a wide variety of restriction enzymes that have been discovered and continue to be discovered

14. Restriction Sites What is a restriction site? What is a restriction site? The area on DNA that the restriction enzyme recognizes The area on DNA that the restriction enzyme recognizes Many different kinds (EcoRI, BamHI, HindIII, etc) Many different kinds (EcoRI, BamHI, HindIII, etc) Like palindrome Like palindrome A palindrome is a word or phrase which reads the same in both directions. A palindrome is a word or phrase which reads the same in both directions. These enzymes make “blunt” and “sticky” ends. These enzymes make “blunt” and “sticky” ends. What is the difference? What is the difference?

16. Restriction Enzymes Enzymes usually make many cuts in a DNA molecule Enzymes usually make many cuts in a DNA molecule Yields a set of restriction fragments Yields a set of restriction fragments Most useful restriction enzymes cut DNA in a staggered way Most useful restriction enzymes cut DNA in a staggered way Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments DNA ligase is an enzyme that seals the bonds between restriction fragments http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# Animation #1 Animation #1

17. Different restriction enzymes that can be used Different restriction enzymes that can be used

19. Recombinant Plasmid Restriction Enzymes: Proteins that cut the DNA in a specific place Gene of interest Cloning Vector

21. Gene Cloning Using a Plasmid The original plasmid is called the cloning vector The original plasmid is called the cloning vector DNA molecule that can carry foreign DNA into a cell and replicate there DNA molecule that can carry foreign DNA into a cell and replicate there After insertion of the gene of interest into the plasmid the plasmid is called recombinant DNA After insertion of the gene of interest into the plasmid the plasmid is called recombinant DNA Once the plasmid is inside the bacterium (host) cell, the cell is called a transformed cell Once the plasmid is inside the bacterium (host) cell, the cell is called a transformed cell

22. REVIEW… How do you clone a gene using a plasmid? How do you clone a gene using a plasmid?

23. Steps in DNA recombination 1. Isolate “needed: DNA gene & plasmid 2. Cut (“digest”) BOTH DNA samples with the SAME restriction enzyme 3. Mix DNA’s together- they join at sticky ends via DNA base pairing 4. Add ligase to seals up sticky ends 5. Product is the recombinant DNA 6. Add CaCl 2 & bacteria CaCl 2 makes bacteria “compentent” 7. Collect products (proteins) of cloned gene (Ex: insulin, Human Growth Hormone)

25. http://highered.mcgraw- hill.com/sites/0072437316/student_view0/chapter16/anim ations.html http://highered.mcgraw- hill.com/sites/0072437316/student_view0/chapter16/anim ations.html http://highered.mcgraw- hill.com/sites/0072437316/student_view0/chapter16/anim ations.html http://highered.mcgraw- hill.com/sites/0072437316/student_view0/chapter16/anim ations.html ANIMATION #8 ANIMATION #8 Plasmid Cloning

26. 3 main reasons for creating recombinant DNA 1. to create a protein product 2. to create multiple copies of genes 3. to insert foreign genes into other organisms to give those organisms a new trait Recombinant DNA is used widely today to create large amounts of protein for treating illnesses Ex: In 1982, insulin became the first recombinant DNA drug to hit the market Why make DNA recombinants?

28. http://www.sumanasinc.com/webcontent/animations/con tent/plasmidcloning.html http://www.sumanasinc.com/webcontent/animations/con tent/plasmidcloning.html http://www.sumanasinc.com/webcontent/animations/con tent/plasmidcloning.html http://www.sumanasinc.com/webcontent/animations/con tent/plasmidcloning.html Plasmid Cloning