G e n e t i c s N e w s

1. GeneticsNews • Seminar Monday, Oct 30John RyanImmunology, asthma • Printing problems

2. Topics for today Phenotypes of strains diploid for lac Model for positive regulation of lac operon Experimental test of model

3. Predict the Lac phenotype ß-galactosidase phenotypeI inducible by IPTGC constitutive activityN no activity GrowthLac+can grow on lactoseLac- cannot grow on lactose Chromosome PlasmidPhenotype I+ P+ O+ Z+ Y+ I+ P+ O+ Z+ Y+

4. Predict the Lac phenotype ß-galactosidase phenotypeN no activity (or very low) - lactose X - lacZ is dead + lactose X X

5. Predict the Lac phenotype ß-galactosidase phenotypeN no activity (or very low) - lactose - lacZ is dead X - promoter is dead + lactose X X X X X

6. Predict the Lac phenotype + + ß-galactosidase phenotypeN no activity - lactose - lacZ is dead- promoter is dead + + lactose I inducible by IPTG - good lacZ, promoter - good lacI + - good operator

7. Predict the Lac phenotype ß-galactosidase phenotypeN no activity X - lacZ is dead- promoter is dead X + I inducible by IPTG - good lacZ, promoter- good lacI- good operator + C constitutive activity - good lacZ, promoter - bad lacI - OR bad operator

8. Predict the Lac phenotype GrowthLac+ can grow on lactose - good lacZ - AND good lacY lacZ+ lacY+

9. Predict the Lac phenotype GrowthLac+ can grow on lactose - good lacZ - AND good lacY X X Lac-cannot grow on lactose - bad lacZ lacZ-

10. Predict the Lac phenotype GrowthLac+ can grow on lactose - good lacZ - AND good lacY X X X Lac-cannot grow on lactose - bad lacZ - OR bad lacY lacY-

11. Lac Phenotype: Strategy Check trans-acting regulatory factors • Repressor • CAP • RNA polymerase Check cis-acting regulatory factors What’s a “trans-acting” factor?What’s a “cis-acting” factor? SQ8. Does a start codon mutation act in cis or trans? SQ9. Would a promoter mutation act in cis or trans? • Promoter • Operator

12. Lac Phenotype: Strategy I- P+ O+ Z+ Y+ F’( I+ P+ Oc Z- Y+) Check trans-acting regulatory factors -+ + + + • Repressor • CAP • RNA polymerase X X Check cis-acting regulatory factors X + +- -+ • Promoter • Operator ß-Gal phenotype? Growth?

13. Lac Phenotype I- P+ O+ Z+ Y+ F’( I+ P+ Oc Z- Y+) ß-Gal phenotype? - lacZ repressed (lacY constitutive) - - Without lactose

14. Lac Phenotype I- P+ O+ Z+ Y+ F’( I+ P+ Oc Z- Y+) ß-Gal phenotype? - lacZ induced (lacY constitutive) INDUCIBLE Growth phenotype? ß-gal made - - Permease made GROWTH With lactose

15. Positive Regulation of lac operon State • Plenty of glucose Response • Eat it • Avoid other sugars Symbol of state • Low cAMP glucose lactose other sugars

16. Positive Regulation of lac operon State • No glucose Response • Eat other sugars Symbol of state • High cAMP lactose other sugars

17. Positive Regulation X X crp- Test of modelWhat happens if... X X Low expression of lac operon

18. Positive Regulation X Test of modelWhat happens if... X X Low expression of lac operon (Why defects lead to low expression?)

19. Positive Regulation X Test of modelWhat happens if... X X X Low expression of lac operon

20. Positive Regulation Protein Regulation Target Acts on Influenced by Lac repressor CRP Negative operator lac operon lactose Positive CRPbinding site many operons cAMP

21. Positive RegulationSQ14: Superimpose cAMP & ß-gal on growth curve When is glucose present? - glucose [ß-gal] When is cAMP present? [cAMP] When is ß-gal present? + glucose

22. Test of Positive Regulation - SQ15 Absence of inducer, ß-gal activity…? Presence of IPTG, ß-gal activity…? Presence of IPTG and glucose, ß-gal activity…? Grown in lactose + glucose, diauxic effect…?

23. Test of Positive Regulation SQ16Is diauxic growth apparent? SQ17Is appearance of ß-gal repressed by glucose? From Kimata et al (1997)

24. SQ22: How to make biosensor of heavy metals Red on MacConkey plates