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Bacterial Transformation What is it? http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/3.1.2_bacteria_3.png Bacterial Transformation What is it? How does it work? http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/3.1.2_bacteria_3.png Bacterial Transformation What is it? How does it work? Where is it used in industry? http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/3.1.2_bacteria_3.png Bacterial Transformation What is it? How does it work? Where is it used in industry? Where can it be used In the future? http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/3.1.2_bacteria_3.png What is it? • Bacterial transformation is a technique used to insert foreign DNA into bacterial cells resulting in new phenotypic expressions. http://www.sciencebuddies.org/science-fairprojects/project_ideas/BioChem_p024.shtml What is it? • Bacterial transformation is a technique used to insert foreign DNA into bacterial cells resulting in new phenotypic expressions. There are two types of transformation http://www.sciencebuddies.org/science-fairprojects/project_ideas/BioChem_p024.shtml What is it? • Bacterial transformation is a technique used to insert foreign DNA into bacterial cells resulting in new phenotypic expressions. There are two types of transformation 1) Natural Transformation http://www.sciencebuddies.org/science-fairprojects/project_ideas/BioChem_p024.shtml What is it? • Bacterial transformation is a technique used to insert foreign DNA into bacterial cells resulting in new phenotypic expressions. There are two types of transformation 1) Natural Transformation 2) Artificial Transformation http://www.sciencebuddies.org/science-fairprojects/project_ideas/BioChem_p024.shtml What is it? • Bacterial transformation is a technique used to insert foreign DNA into bacterial cells resulting in new phenotypic expressions. There are two types of transformation 1) Natural Transformation 2) Artificial Transformation Escherichia coli HB101 transformed using pGLO plasmid http://www.sciencebuddies.org/science-fairprojects/project_ideas/BioChem_p024.shtml Natural Bacterial Transformation • Certain species of bacteria are capable of accepting foreign DNA and utilizing the DNA to produce new traits. This is known as natural competence. http://www.cdc.gov/hi-disease/about/photos.html https://en.wikipedia.org/wiki/File:Bacillus_subtilis_colonies.jpg http://academic.pgcc.edu/~kroberts/web/colony/spnuemo.gif https://en.wikipedia.org/wiki/File:Neisseria_gonorrhoeae_Growth_on_New_York_City_Agar_Plate.jpg http://www.allposters.com/-sp/Streptococcus-Pneumoniae-Bacteria-Gram-Positive-Cocci-Showing-Capsules-Posters_i9005107_.htm Natural Bacterial Transformation • Certain species of bacteria are capable of accepting foreign DNA and utilizing the DNA to produce new traits. This is known as natural competence. Bacillus subtilis http://www.cdc.gov/hi-disease/about/photos.html https://en.wikipedia.org/wiki/File:Bacillus_subtilis_colonies.jpg http://academic.pgcc.edu/~kroberts/web/colony/spnuemo.gif https://en.wikipedia.org/wiki/File:Neisseria_gonorrhoeae_Growth_on_New_York_City_Agar_Plate.jpg http://www.allposters.com/-sp/Streptococcus-Pneumoniae-Bacteria-Gram-Positive-Cocci-Showing-Capsules-Posters_i9005107_.htm Natural Bacterial Transformation • Certain species of bacteria are capable of accepting foreign DNA and utilizing the DNA to produce new traits. This is known as natural competence. Bacillus subtilis Streptococcus pneumoniae http://www.cdc.gov/hi-disease/about/photos.html https://en.wikipedia.org/wiki/File:Bacillus_subtilis_colonies.jpg http://academic.pgcc.edu/~kroberts/web/colony/spnuemo.gif https://en.wikipedia.org/wiki/File:Neisseria_gonorrhoeae_Growth_on_New_York_City_Agar_Plate.jpg http://www.allposters.com/-sp/Streptococcus-Pneumoniae-Bacteria-Gram-Positive-Cocci-Showing-Capsules-Posters_i9005107_.htm Natural Bacterial Transformation • Certain species of bacteria are capable of accepting foreign DNA and utilizing the DNA to produce new traits. This is known as natural competence. Bacillus subtilis Streptococcus pneumoniae Neisseria gonorrhoeae http://www.cdc.gov/hi-disease/about/photos.html https://en.wikipedia.org/wiki/File:Bacillus_subtilis_colonies.jpg http://academic.pgcc.edu/~kroberts/web/colony/spnuemo.gif https://en.wikipedia.org/wiki/File:Neisseria_gonorrhoeae_Growth_on_New_York_City_Agar_Plate.jpg http://www.allposters.com/-sp/Streptococcus-Pneumoniae-Bacteria-Gram-Positive-Cocci-Showing-Capsules-Posters_i9005107_.htm Natural Bacterial Transformation • Certain species of bacteria are capable of accepting foreign DNA and utilizing the DNA to produce new traits. This is known as natural competence. Bacillus subtilis Neisseria gonorrhoeae Streptococcus pneumoniae Haemophilus influenzae http://www.cdc.gov/hi-disease/about/photos.html https://en.wikipedia.org/wiki/File:Bacillus_subtilis_colonies.jpg http://academic.pgcc.edu/~kroberts/web/colony/spnuemo.gif https://en.wikipedia.org/wiki/File:Neisseria_gonorrhoeae_Growth_on_New_York_City_Agar_Plate.jpg http://www.allposters.com/-sp/Streptococcus-Pneumoniae-Bacteria-Gram-Positive-Cocci-Showing-Capsules-Posters_i9005107_.htm Artificial Bacterial Transformation • Bacteria that are not naturally competent require additional steps to open and depolarize the membranes. https://upload.wikimedia.org/wikipedia/commons/7/73/Ecoli_colonies.png http://www.microbeworld.org/component/jlibrary/?view=article&id=13348 Artificial Bacterial Transformation • Bacteria that are not naturally competent require additional steps to open and depolarize the membranes. An example of a non competent bacteria often used in bacteria transformation is Escherichia coli. https://upload.wikimedia.org/wikipedia/commons/7/73/Ecoli_colonies.png http://www.microbeworld.org/component/jlibrary/?view=article&id=13348 Artificial Bacterial Transformation • Bacteria that are not naturally competent require additional steps to open and depolarize the membranes. An example of a non competent bacteria often used in bacteria transformation is Escherichia coli. 1. To “open up” the membranes and cell wall a bacterial culture is suspended in cold CaCl2. CaCl2 neutralizes the overall negative charge present on the cell surface. https://upload.wikimedia.org/wikipedia/commons/7/73/Ecoli_colonies.png http://www.microbeworld.org/component/jlibrary/?view=article&id=13348 Artificial Bacterial Transformation • Bacteria that are not naturally competent require additional steps to open and depolarize the membranes. An example of a non competent bacteria often used in bacteria transformation is Escherichia coli. 1. To “open up” the membranes and cell wall a bacterial culture is suspended in cold CaCl2. CaCl2 neutralizes the overall negative charge present on the cell surface. 2. Then the culture is heat shocked at around 42oC, then 5oC. This physically opens up micro pores and allows the plasmid to penetrate past the bacterial cell wall and membrane. https://upload.wikimedia.org/wikipedia/commons/7/73/Ecoli_colonies.png http://www.microbeworld.org/component/jlibrary/?view=article&id=13348 Outer Structure of Bacteria http://study.com/cimages/multimages/16/calcium-chloride-solution-sm.jpg https://en.wikipedia.org/wiki/File:Gram_negative_cell_wall.svg https://en.wikipedia.org/wiki/File:Gram-positive_cellwall-schematic.png Outer Structure of Bacteria Calcium chloride binding to the negative phosphate groups on the outer phospholipid bilayer complex. http://study.com/cimages/multimages/16/calcium-chloride-solution-sm.jpg https://en.wikipedia.org/wiki/File:Gram_negative_cell_wall.svg https://en.wikipedia.org/wiki/File:Gram-positive_cellwall-schematic.png Outer Structure of Bacteria Calcium chloride binding to the negative phosphate groups on the outer phospholipid bilayer complex. A sudden increase in temperature creates pores in the plasma membrane of the bacteria http://study.com/cimages/multimages/16/calcium-chloride-solution-sm.jpg and allows for plasmid DNA to enter the https://en.wikipedia.org/wiki/File:Gram_negative_cell_wall.svg bacterial cell. https://en.wikipedia.org/wiki/File:Gram-positive_cellwall-schematic.png Electroporation • Electroporation is the application of an electrical current across a cell membrane resulting in temporary “pore” formation enabling the uptake of exogenous molecules. http://faculty.ccri.edu/lmfrolich/Microbiology/GeneticEngineering.htm Electroporation • Electroporation is the application of an electrical current across a cell membrane resulting in temporary “pore” formation enabling the uptake of exogenous molecules. http://faculty.ccri.edu/lmfrolich/Microbiology/GeneticEngineering.htm https://upload.wikimedia.org/wikipedia/commons/thumb/c/cc/Pore_schematic.svg/578px-Pore_schematic.svg.png Electroporation • Electroporation is the application of an electrical current across a cell membrane resulting in temporary “pore” formation enabling the uptake of exogenous molecules. The electrical current disturbs the phospholipid, it simultaneously allows charged molecules like DNA to be driven across the membrane through the pores in a manner similar to electrophoresis. http://faculty.ccri.edu/lmfrolich/Microbiology/GeneticEngineering.htm https://upload.wikimedia.org/wikipedia/commons/thumb/c/cc/Pore_schematic.svg/578px-Pore_schematic.svg.png Electroporation • Electroporation is the application of an electrical current across a cell membrane resulting in temporary “pore” formation enabling the uptake of exogenous molecules. The electrical current disturbs the phospholipid bilayer, and it simultaneously allows charged molecules like DNA to be driven across the membrane through the pores in a manner similar to electrophoresis. http://faculty.ccri.edu/lmfrolich/Microbiology/GeneticEngineering.htm https://upload.wikimedia.org/wikipedia/commons/thumb/c/cc/Pore_schematic.svg/578px-Pore_schematic.svg.png How does it work? Plasmids http://1.bp.blogspot.com/-bK2ABP_Q3E4/U1axT0nwqoI/AAAAAAAABEY/fJpYm6IfFHs/s1600/Lab7-B1.jpg How does it work? Plasmids • A plasmid is a small circular double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. http://1.bp.blogspot.com/-bK2ABP_Q3E4/U1axT0nwqoI/AAAAAAAABEY/fJpYm6IfFHs/s1600/Lab7-B1.jpg How does it work? Plasmids • A plasmid is a small circular double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. • Plasmids can replicate independently from the host’s DNA and often provide bacteria with genetic advantages, such as antibiotic resistance. http://1.bp.blogspot.com/-bK2ABP_Q3E4/U1axT0nwqoI/AAAAAAAABEY/fJpYm6IfFHs/s1600/Lab7-B1.jpg How does it work? Plasmids • A plasmid is a small circular double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. • Plasmids can replicate independently from the host’s DNA and often provide bacteria with genetic advantages, such as antibiotic resistance. pGLO plasmid with origin, arabinose operon, ampicillin resistance gene and restriction enzyme cut sites. http://1.bp.blogspot.com/-bK2ABP_Q3E4/U1axT0nwqoI/AAAAAAAABEY/fJpYm6IfFHs/s1600/Lab7-B1.jpg Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. Electron micrograph of plasmid http://www.pnas.org/content/110/39.cover-expansion Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. • Approximately 1 in 10,000 bacteria will succeed in plasmid incorporation. Electron micrograph of plasmid http://www.pnas.org/content/110/39.cover-expansion Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. • Approximately 1 in 10,000 bacteria will succeed in plasmid incorporation. Electron micrograph of plasmid http://www.pnas.org/content/110/39.cover-expansion Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. • Approximately 1 in 10,000 bacteria will succeed in plasmid incorporation. Electron micrograph of plasmid Bacterial selection http://www.pnas.org/content/110/39.cover-expansion Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. • Approximately 1 in 10,000 bacteria will succeed in plasmid incorporation. Electron micrograph of plasmid Bacterial selection http://www.pnas.org/content/110/39.cover-expansion Start of replication Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. • Approximately 1 in 10,000 bacteria will succeed in plasmid incorporation. Electron micrograph of plasmid Gene of interest Bacterial selection http://www.pnas.org/content/110/39.cover-expansion Start of replication Plasmids • Once the Plasmid is successfully in the bacterial cytoplasm, it migrates to the circular DNA and incorporates into the genome. • Approximately 1 in 10,000 bacteria will succeed in plasmid incorporation. Electron micrograph of plasmid Gene promoter Gene of interest Bacterial selection http://www.pnas.org/content/110/39.cover-expansion Start of replication Results • Bacteria that have been successfully transformed will express the genes that the plasmid coded for. https://b51ab7d9e5e1e7063dcb70cee5c33cf7f4b7bad8.googledrive.com/host/0Bx6hk6AUBHxDc2d4TDJZTFIyMGs /files/Bio%20101/Bio%20101%20Laboratory/Bacterial%20Transformation/results.htm Results • Bacteria that have been successfully transformed will express the genes that the plasmid coded for. • In most cases there is a selective marker in which the transformed bacteria can withstand certain antibiotics. https://b51ab7d9e5e1e7063dcb70cee5c33cf7f4b7bad8.googledrive.com/host/0Bx6hk6AUBHxDc2d4TDJZTFIyMGs /files/Bio%20101/Bio%20101%20Laboratory/Bacterial%20Transformation/results.htm Results • Bacteria that have been successfully transformed will express the genes that the plasmid coded for. • In most cases there is a selective marker in which the transformed bacteria can withstand certain antibiotics. • This antibiotic marker allows researchers to isolate transformed colonies. https://b51ab7d9e5e1e7063dcb70cee5c33cf7f4b7bad8.googledrive.com/host/0Bx6hk6AUBHxDc2d4TDJZTFIyMGs /files/Bio%20101/Bio%20101%20Laboratory/Bacterial%20Transformation/results.htm How are commercial plasmids engineered? • 1. Bacterial plasmid isolated, then treated with restriction enzymes to cut a specific band pattern and length How are commercial plasmids engineered? • 1. Bacterial plasmid isolated, then treated with restriction enzymes to cut a specific band pattern and length • 2. Foreign DNA is cut using restriction enzymes with the same band pattern. This produces “sticky ends” for the plasmid and DNA sequence to connect. How are commercial plasmids engineered? • 1. Bacterial plasmid isolated, then treated with restriction enzymes to cut a specific band pattern and length • 2. Foreign DNA is cut using restriction enzymes with the same band pattern. This produces “sticky ends” for the plasmid and DNA sequence to connect. • 3. DNA ligase repairs the cuts and the new plasmid is stored in a dry freeze, lyophilization. Current Applications • Current applications: Current Applications • Current applications: • production of insulin Current Applications • Current applications: • production of insulin • vaccines Current Applications • • • • Current applications: production of insulin vaccines antibiotics Current Applications • • • • • Current applications: production of insulin vaccines antibiotics other specific human proteins. Current Applications • • • • • Current applications: production of insulin vaccines antibiotics other specific human proteins. Prior to bacterial insulin production approximately 2 tons of pig tissue was used to produce 8 ounces of purified insulin. Current Applications • • • • • Current applications: production of insulin vaccines antibiotics other specific human proteins. Prior to bacterial insulin production approximately 2 tons of pig tissue was used to produce 8 ounces of purified insulin. Future Applications Microbes Here to Clean Up Our Environmental Messes • Future applications in bioremediation are currently being researched to clean up crude oil spills and mercury contamination. Alcanivorax borkumensis degrading oil Microbes, the worlds unlimited renewable resource Microbes, the worlds unlimited renewable resource Oil decomposition Alcanivorax borkumensis Microbes, the worlds unlimited renewable resource Oil decomposition Alcanivorax borkumensis Mercury detoxification Escherichia coli Microbes, the worlds unlimited renewable resource Oil decomposition Escherichia coli Alcanivorax borkumensis Polyethylene decomposition Enterobacter asburiae YT1 Mercury detoxification Microbes, the worlds unlimited renewable resource Oil decomposition Mercury detoxification Escherichia coli Alcanivorax borkumensis Polyethylene decomposition Enterobacter asburiae YT1 Isoprene Production Escherichia coli Review • What is it? Incorporation of foreign DNA into bacterial cells • How does it work? Plasmid incorporation, CaCl2-heat shock, electroporation. • Where is it used in industry? Production of insulin and human proteins • Where can it be used In the future? Bioremediation Thank you