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AP BIOLOGY ACTIVITY: BUILDING PROKARYOTIC OPERONS Assignment For Table Groups 1-5: Your Group of 3 will build a negative REPRESSIBLE operon (trp). Your model should include representations the following elements: • • • • • • A constitutive Repressor Gene located “some distance” from your operon A promoter region that allows the binding of RNA Polymerase An Operator Region that allows the binding of the repressor protein The Structural Genes that comprise the rest of the operon A repressor Protein with an active AND an allosteric site A small regulatory molecule capable of causing a conformational shape change in the repressor protein Instructions: 1. Using a serrated knife, cut an 8-inch segment from the noodle. This segment will be used as the repressor protein. 2. Each end of the noodle/operon should feature an unlabeled/untaped section to show the continuation of the DNA strand. 3. Wrap spirals of colored electrical tape (or shade the noodle with colored markers) where each of the 5 gene domain regions would be found (trp E, trp D, trp C, trp B , and trp A), using a different color for each gene domain. 4. Tape or shade in the regulatory gene (trp R) region as far upstream of the promoter region as possible. 5. Using a permanent pen, draw the shape of the active form of the repressor protein onto the lower portion of the noodle/operon, in the operator region. Make the shape simple since you will need to carve it out using a serrated knife. Also, carve a matching shape into the regulatory repressor protein piece that you cut off in step 1 above. 6. On the bottom side of the repressor protein, carve a U and wedge the tennis ball into the U. 7. Cut a piece of wire from a coat hanger and shove the wire into the repressor protein and bend the repressor protein into a shape so that it will not fit the operator region if the co-repressor (tryptophan) is not in place. 8. Write the word “tryptophan” on one of the tennis balls. Write “repressor protein” on the carved foam piece. Now label the various parts of the noodle/operon using a permanent marker “trp R,” “promoter/operator,” “trp E,” “trp D,” “trp C,” “trp B,” and “trp A.” 9. Place stick-on tabs on the parts of the operator and the repressor protein that fit together, so that they can stick together without being held in place. You may do the same for the repressor and the co-repressor/tryptophan ball. Assignment For Table Groups 6-10: Your group of 3 will build a functioning operon that is capable of demonstrating positive control (Lac operon). Your model should include representations the following elements: • A constitutive Repressor Gene located “some distance” from your operon • A promoter region that allows the binding of RNA Polymerase • An Operator Region that allows the binding of the repressor protein • The Structural Genes that comprise the rest of the operon • A repressor Protein with an active AND an allosteric site • A small regulatory molecule capable of causing a conformational shape change in the repressor protein • CAP and CAP binding Site Instructions: 1. Using a serrated knife, cut an 8-inch segment from the noodle. This will be used as the repressor protein. 2. Again, each end of the noodle/operon should feature an unlabeled/untaped section, to show the continuation of the DNA strand. 3. Wrap spirals of colored electrical tape (or shade the noodle with colored markers) where each of the 3 gene domain regions would be found (lac Z, lac Y, and lac A), using a different color for each gene domain. 4. Tape or shade in the regulatory gene (lac I) region, which is immediately upstream of the promoter region. 5. Using a permanent pen draw the shape of the active form of the repressor protein onto the lower portion of the noodle in the operator region. Make the shape simple, like the one in the diagram, since you will need to carve it out using a serrated knife. Also, carve a matching shape into the regulatory repressor protein piece that you cut off in step 1 above. 6. On the bottom side of the repressor protein, carve a wide, semicircle shape that is a little too wide to accommodate the tennis ball. You want the repressor protein to have 2 shapes, one that fits the operator shape perfectly when the inducer is NOT present and one that distorts the repressor so that the carved top shape appears to pop out of the operator when the inducer fits into the bottom (you can shove a piece of coat hanger wire into the repressor to make it hold 2 different shapes). 7. Write “allolactose” on one of the tennis balls. Write “repressor protein” on the carved foam piece. Write “lac I,” “promoter/operator,” “lac Z,” “lac Y,” and “lac A” at the appropriate places along the noodle. 8. You may place stick-on tabs on both the operator and repressor protein parts so that they can stick together without being held in place. You may do the same for the repressor and the corepressor/allolactose ball. ALL TABLE GROUPS: Once the Operon Models have been constructed, Table groups will pair up as follows: 1+6 2+7 3+8 4+9 5 +10 Each group needs to prepare and practice a short verbal presentation using the operon models your group has built as a prop. Your presentation needs to be able to: (a) explain how the operon works within the nutritional circumstances it is likely to find itself (b) distinguish between Negative and Positive control (c) distinguish between Inducible and Repressible operons. Each group will receive a grade that is based on 3 criteria: 1. Accuracy of model 2. Thoroughness of explanation 3. Your table mate’s grades on a short quiz. (In other words, you will be grades on the success of your students!)