Modeling Plasmid Selection

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Modeling Plasmid Selection
Joy Killough RET Teacher University of Texas at
Austin With Dr. C. Randall Linder
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Modeling Plasmid Selection

• Bacteria are very useful organisms in genetic
  engineering. They are able to bring in plasmids
  into which DNA of interest may be added.
• Being able to find the bacteria containing the
  plasmid that has been modified requires some
  techniques that look for the effects of plasmid
  insertion, which are visible to the naked eye,
  rather than looking for the plasmid itself (which
  of course is not).

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Materials Needed

• For each student group provide
• Four ½ sheets of paper cut into ovals to
  represent bacteria
• Four rings of pony beads, each on a full chenille
  stem (pipe cleaner), formed into a circle to
  represent a chromosome. Group several beads of a
  color together to represent genes.
• Three smaller rings of beads (half of a stem) to
  represent plasmids. Make them identical and be
  sure they have a pair of purple beads (antibiotic
  resistance gene). Let the first and last bead be
  green ( lac Z gene) and close into a circle
  leaving an inch or so of the stem at each end
  without beads.
• 2 pink beads representing the foreign gene of
  interest that is being added to the plasmid.

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Restriction Enzymes

• Restriction enzymes cut DNA at very specific
  locations. They are very predictable, each
  enzyme always cutting the same way. This
  characteristic is used in genetic engineering.
• Plasmids are cut with the same restriction enzyme
  used to cut the DNA to be inserted. A
  restriction enzyme which leaves overhanging
  sticky ends is needed for this this procedure.
  This provides the free base pairs for combining
  the plasmid DNA with the source DNA.

Restriction Enzyme Cut from EcoRI
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Modeling the plasmid

• To represent a plasmid, make a small ring of
  about 10 pony beads strung on a chenille stem
  (pipe cleaner). Include 2 purple beads to
  represent the antibiotic resistance gene (ampr)
  and 2 green beads on each side of the closure to
  represent the gene Lac Z. Lac Z codes for beta
  galactosidase, the protein that can cleave the
  sugar, X-gal, to make a blue colony. Each group
  of beads of a single color represents a gene.

Gene for antibiotic resistance
LacZ
Restriction site
Tails of chenille stems
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• Provide three plasmid bead rings and two DNA
  pieces (pink beads) per student group. Instruct
  students to add the genes to the plasmids at the
  restriction site. It is deliberate that they
  have fewer gene beads than plasmids.

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Modeling the plasmid cut with restriction enzymes

• Tell students to open the plasmid by untwisting
  the chenille stem. This represents the
  recognition site for the restriction enzyme. Add
  a pink bead to represent the gene of interest
  that is now being added to the plasmid since it
  shares the same sticky ends as the plasmid.

Gene for antibiotic resistance
Restriction Site
LacZ
Tails of chenille stems
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Cut 4 ovals the size of a half a sheet of paper.
Each represents a single bacterial cell. Have
each student group lay these out on a large
surface. Prepare 4 large rings of pony beads
strung on pipe cleaners (chenille stems).
Sections of beads of a single color represent
various genes and the entire ring represents the
prokaryotic chromosome. Students should place
the chromosome inside the bacterial cell.
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Tell students to imagine the area around the
bacteria is a Petri dish with nutrient agar. Ask
them If each of these four bacteria were placed
on the Petri dish how many would live ? Student
should answer all four. Ask them What would the
plate look like? Students should realize each
bacteria would form a small white colony. Point
out this contains thousands and thousands of
bacteria, clones of the original.
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Transformation
To model transformation have students place their
modified plasmids into the bacteria. Ask them
Which bacteria would survive on a plate of
nutrient agar? Again they should choose all four.
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Selection for Antibiotic Resistance
Now have students imagine the area around the
bacteria is a Petri dish with nutrient agar with
ampicillin and the sugar X-gal added. Ask them
If each of these four bacteria were placed on the
Petri dish how many would live ? This time they
should choose three. Only the bacteria with the
antibiotic resistance gene (purple) in the
plasmid present will survive to form a colony.
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Selection for breakdown of X-gal
Of the surviving colonies which will be white and
which blue? Ask the students why. Answer The
insertion of the pink gene (the gene of interest)
into the green LacZ gene breaks the gene so it
can no longer cleave X-gal forming the blue
colony. Which colony contain the inserted gene
pieces? The white colonies.
ampr
lacZ
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Modeling Plasmid SelectionStudent Questions

• 1. How are prokaryotic chromosomes and eukaryotic
  chromosomes different?
• 2. What is a plasmid?
• 3. How is a plasmid different from a chromosome?
• 4. Why are indirect means used to identify the
  presence of an inserted piece of DNA in a
  plasmid?
• 5. How do restriction enzymes cut?
• 6. What are sticky ends and why are they so
  important?
• 7. What procedure makes it likely that a plasmid
  and a piece of foreign DNA will combine?
• 8. Research the restriction enzyme and find the
  difference between a blunt cut and a cut which
  leaves sticky ends.
• 9. Research the restriction enzyme and find out
  how they are named.
• 10. Research the discovery of restriction
  enzymes.