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

2. 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).

3. 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.

4. 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

5. 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 (amp r ) 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. Tails of chenille stems Gene for antibiotic resistance LacZ’Restriction site

6. 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.

7. 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. Tails of chenille stems Gene for antibiotic resistance LacZ’ Restriction Site

8. 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.

9. 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.

10. 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.

11. 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.

12. 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. amp r lacZ’

13. Modeling Plasmid Selection Student 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.