Genetics News Seminar Monday Oct 30 John Ryan

Genetics News • Seminar Monday, Oct 30 John Ryan Immunology, asthma • Printing problems

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

Predict the Lac phenotype ß-galactosidase phenotype I inducible by IPTG C constitutive activity N no activity Growth Lac+ can grow on lactose Lac- cannot grow on lactose Chromosome I+ P+ O+ Z+ Y+ Plasmid I+ P+ O+ Z+ Y+ Phenotype

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

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

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

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

Predict the Lac phenotype Growth Lac+ can grow on lactose - good lac. Z - AND good lac. Y+ lac. Z+

Predict the Lac phenotype X lac. Z- Growth Lac+ can grow on lactose - good lac. Z - AND good lac. Y X Lac- cannot grow on lactose - bad lac. Z

Predict the Lac phenotype Growth Lac+ can grow on lactose - good lac. Z - AND good lac. Y X X lac. Y- X Lac- cannot grow on lactose - bad lac. Z - OR bad lac. Y

Lac Phenotype: Strategy Check trans-acting regulatory factors • Repressor • CAP • RNA polymerase Check cis-acting regulatory factors SQ 8. SQ 9. Does Would a start a promoter codon mutation act What’s a “trans-acting” factor? in cis or trans? What’s a “cis-acting” factor? • Promoter • Operator

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

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

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

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

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

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

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

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

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

Positive Regulation SQ 14: Superimpose c. AMP & ß-gal on growth curve When is glucose present? - glucose [ß-gal] [c. AMP] When is c. AMP present? When is ß-gal present? + glucose

Test of Positive Regulation - SQ 15 Absence of inducer, ß-gal activity…? Presence of IPTG and glucose, ß-gal activity…? Grown in lactose + glucose, diauxic effect…?

Test of Positive Regulation SQ 16 Is diauxic growth apparent? SQ 17 Is appearance of ß-gal repressed by glucose? From Kimata et al (1997)

SQ 22: How to make biosensor of heavy metals Red on Mac. Conkey plates