Plasmids and Cloning DNA by hcj


									        What is DNA Cloning?
• A fragment of DNA that has been introduced
  into a foreign cell
• This results in exact copies of the original
  DNA fragment being made when the foreign
  cell replicates and divides
• Gene cloning became possible after Watson,
  Crick and other researchers began to
  discover the way molecules transmit genetic
• In the 1970’s, this technique
             What Are Plasmids?
• Genetic structures which have the ability
  to replicate independently of the
  chromosomes (typically a small circular DNA strand in the
  cytoplasm of a bacterium or protozoan)

• They possess a copy number- # of copies
  of particular plasmid found in a bacterial
 Restriction Endonucleases (RE)
• Enzymes which slice DNA into fragments at
  specific sequences (4-8 base pairs long)
• When sliced at the ends, the DNA becomes
  “sticky” if there is a single-strand exposed
• Certain R E need to be used to ensure the gene
  being analyzed is removed completely from the
• Multiple R E are being used at the same time
      How do you Separate the Wanted
     Fragments with the Unwanted Ones?
• Through a process called gel
• This process causes the
  separation of charged fragments
  of DNA (due to negative charge
  on phosphate group) on the
  basis of size by sorting through a
  gel meshwork. Shorter
  fragments travel the fastest,
  longer ones travel slowest
      Recombinant DNA Molecule
  Think of a puzzle piece…
• The plasmids must also be cleaved using the same
  restriction endonuclease that cleaved the desired gene.
  The fragments of DNA that were cut are now joined
  (ligated) to the plasmid and is able to fit perfectly due to
  each of their “sticky” ends, which will match up like a
• Plasmids are examples of “DNA vectors” which are
  vehicles by which DNA is brought into host cells
• The plasmids can now replicate independently in a host
• The combination between the DNA fragments and the
  plasmids is called   recombinant
For Example…
        • In this scenario,
          EcoR1 is the
          endonuclease that
          is responsible for
          the cleaving of both
          the plasmid and the
        • This diagram
          portrays the
          construction of the
          recombinant DNA
        • Notice the overhang
          of the DNA
          fragments that fit
          perfectly into the
          overhang of the
   How is the Recombinant DNA
   Introduced into the Host Cell?
• Bacterial host cells can be manipulated in
  order to host the recombinant DNA
• This is done through the use of gene guns,
  electroporators (chambers that subject the
  bacteria to electric shock), or classical
  transformation protocols, such as calcium
• The bacteria is considered
 transformed when it takes on the
 recombinant DNA
     How does the Calcium Chloride
           Protocol Work?
• Bacteria can become competent, or ready to take up
  foreign DNA, through the use of calcium chloride
• Calcium chloride ionizes into + and - ions, and the
  bacterial cell membranes are frozen (become rigid) by
  being submerged into a 0°C solution of the CaCl2
• Negatively charged phosphates in membrane are
  stabilized by the positively charged calcium ions- now
  stabilized physically and chemically
• Plasmid DNA is introduced into the solution
• The solution undergoes a quick heat shock treatment of
  42°C for 90 seconds- this creates a draft and plasmids
  are swept through membrane’s pores
• Bacteria recovers- incubated in a media suspension full
   How are the Transformed
       Cells Selected?

• Need to yield successfully transformed
  cells with recombinant DNA
• These cells are chemically selected by the
  presence of a marker on the plasmid (one
  example of this is antibiotic resistance)
           Selective Plating
• Plasmids carry an antibiotic-resistant gene
• If the transformation is a success, the
  bacteria will grow on a media containing the
• If no growth is observed, bacteria were not
  transformed and were destroyed by antibiotic
• Positive and negative controls are necessary
  to see how valid your selective plating is
                 What’s Next?
• It is necessary to check if the foreign gene is present in
  the transformation as the bacteria may be antibiotic
  resistant by taking up the plasmid that has failed to have
  the foreign gene fragment inserted
• Individual colonies are removed from a colony that has
  undergone transformation and allowed to increase
  quickly in a liquid media until enough can be
  accumulated to extract a suitable amount of plasmid
• Plasmid DNA is subjected to a restriction enzyme
  digestion to release cloned sequence from plasmid
• If expected pattern of bands is observed on the gel, then
  the colony possesses a recombinant DNA plasmid
         Medical Applications
• Certain diseases that are caused by faulty
  genes, such as sickle-cell anemia, Tay-Sachs
  disease and cystic fibrosis can potentially be
  cured in the future (this is called gene therapy)
  thanks to recombinant DNA technology
• Insulin could be created using gene cloning,
  eliminating the need for the use of animal insulin
  for diabetics- it is now less expensive and more
• Hereditary diseases are caused by damaged, or
  mutated, genes and through testing of samples
  of recombinant DNA cloning, scientists can
          Microbial Cells
• One of the best known uses for
  engineered microbes is as bacteria
  that eat spilled crude oil
• The genetic sequence of the
  microbes can be modified using
  recombinant techniques
• Large amounts of microbes can then
           Ice-Minus Pseudomonas
              Syringae Bacteria
•   Ice-minus Pseudomonas Syringae bacteria is an example of a real-world
•   P.Syringae, or Ice-Major, dwells on the leaves of plants and it possesses a
    gene that creates a protein that causes the leaves of the plant to become an
    ideal surface for ice to form, which will damage the crops
•   There is also a different strain of these bacteria that cannot create this
    protein, known as Ice-Minus Pseudomonas Syringae
•   The gene that produces the unwanted protein is cleaved out using
    restriction endonucleases, then is deactivated in a labratory
•   It is then inserted into a plasmid and multiple copies of the gene are made
•   Then using PCR and UV light, Ice-Minus is created and sprayed on crops
•   Assists in keeping crops alive during cold weather
          The End…?
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