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Bacterial Transformation. Green Fluorescent Protein. What is Transformation?. Change… In molecular biology, change in which genetic material carried by an individual cell is altered by incorporation of foreign DNA. Applications.

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Bacterial transformation

Bacterial Transformation

Green Fluorescent Protein


What is transformation
What is Transformation?

  • Change…

  • In molecular biology, change in which genetic material carried by an individual cell is altered by incorporation of foreign DNA


Applications
Applications

  • Genes coding for traits such as disease or pest resistance may be transformed into plants

  • Gene therapy replaces defective genes in a cell with functional copies

  • Bacteria can be transformed with a gene for human hormone like insulin so it can be produced in mass quantities


Organism to be transformed e coli
Organism to be Transformed: E. coli

  • E. coli bacteria are part of the normal flora in the human gut

  • MM294 is a “K-12” strain that does not colonize the gut

  • Proper decontamination is required nevertheless


Gene to insert green fluorescent protein
Gene to Insert:Green Fluorescent Protein

  • Comes from bioluminescent jellyfish -Aequorea victoria

  • GFP is widely used as a marker to determine if transformation is effective


Gene to insert antibiotic resistance
Gene to Insert:Antibiotic Resistance

  • Beta-lactamase which provides resistance to ampicillin

  • Inactivates ampicillin and allows bacterial growth

  • Also used as a marker


Plasmid
Plasmid

  • A plasmid is a small piece of circular DNA in bacteria in addition to bacterial chromosome

  • Typically contain genes for traits beneficial for survival such as antibiotic resistance

  • In nature, bacteria transfer these plasmids back and forth

  • Acts as a vector – a gene taxi to get new DNA into bacteria


Constructing a plasmid
Constructing a Plasmid

  • Specific fragments of DNA may be isolated, cut into discrete pieces by the action of restriction enzymes, and rejoined by the action of DNA ligase

    http://www.dnai.org/b/index.html


Recombinant

Plasmid

E. coli host cell

Transformed Bacteria


Transformation methods
Transformation Methods

Agrobacterium tumefaciens

  • Naturally occurring soil bacteria that causes Crown Gall disease

  • Ability to infect a plant cell and transfer its DNA

  • Disease causing genes removed and gene of interest inserted


Transformation methods1
Transformation Methods

Biolistics

  • “Gene gun”

  • DNA stuck to microscopic gold or tungsten particles

  • Blast of pressurized helium shoots DNA into plant tissue, some makes it through plant cell walls and membranes and into the nucleus


Transformation methods2
Transformation Methods

Additional Techniques

  • Electroporation

  • Heat Shock


Steps in this lab
Steps in This Lab

  • We will be using a plasmid construct that already contains our gene of interest

  • Prepare cells to allow transfer of DNA through membrane

    • Add ++ ions

    • Heat shock

  • Provide nutrients and allow cells to express newly inserted genes

  • Grow transformed cells on agarose plates to isolate transformants



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