Transcript Bacterial Transformation

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