1. Genetics NewsGenetics News Seminar Monday, Oct 30 John Ryan Immunology, 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 Chromosome Plasmid Phenotype I+ P+ O+ Z+ Y+ ß-galactosidase phenotype I inducible by IPTG C constitutive activity N no activity Growth Lac + can grow on lactose Lac - cannot grow on lactose

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

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

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

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

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

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

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

11. Lac Phenotype: Strategy Check trans-acting regulatory factors Repressor CAP RNA polymerase Check cis-acting regulatory factors Promoter Operator 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?

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

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

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

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

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

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

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

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

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

21. Positive Regulation SQ14: Superimpose cAMP & ß-gal on growth curve When is glucose present? + glucose - glucose When is cAMP present? [cAMP] When is ß-gal present? [ß-gal]

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 From Kimata et al (1997) SQ16 Is diauxic growth apparent? SQ17 Is appearance of ß-gal repressed by glucose?

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