Genetics News• Seminar Monday, Oct 30

John RyanImmunology, 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

Chromosome Plasmid Phenotype

I+ P+ O+ Z+ Y+ 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

Predict the Lac phenotype

ß-galactosidase phenotype N no activity (or very low)

- lacZ is deadX

X

X

- lactose

+ lactose

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

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

++

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

+

+

XX

Predict the Lac phenotype

Growth Lac+ can grow on lactose

- good lacZ - AND good lacY

lacZ+

lacY+

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

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

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?

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+ Oc Z- Y+)

- + + + +

+ + - - +

X

X

X

ß-Gal phenotype? Growth?

Lac Phenotype

I- P+ O+ Z+ Y+ F’( I+ P+ Oc Z- Y+)

-

- -

ß-Gal phenotype?

Without lactose

lacZ repressed

(lacY constitutive)

Lac Phenotype

I- P+ O+ Z+ Y+ F’( I+ P+ Oc Z- Y+)

-

- -

ß-Gal phenotype?

With lactose

lacZ induced

(lacY constitutive)

INDUCIBLE

Growth phenotype?

ß-gal made

Permease made

GROWTH

Positive Regulation of lac operon

glucose lactose other sugars

State

Response

• Avoid other sugars

• Plenty of glucose

• Eat it

Symbol of state• Low cAMP

Positive Regulation of lac operon

lactose other sugars

State

Response

• No glucose

• Eat other sugars

Symbol of state• High cAMP

Positive Regulation

X

Low expression of lac operon

Test of modelWhat happens if...

X X

X

crp-

Positive Regulation

X

X

Test of modelWhat happens if... X

Low expression of lac operon(Why defects lead to low expression?)

Positive Regulation

XX

Test of modelWhat happens if... X

Low expression of lac operon

X

Positive Regulation

Protein Regulation Target Acts on Influenced byLac repressor

CRP

Negative operator lac operon lactose

Positive CRPbinding site

many operons cAMP

Positive RegulationSQ14: Superimpose cAMP & ß-gal on growth curve

When is glucose present?

+ glucose

- glucoseWhen is cAMP present?[cAMP]

When is ß-gal present?

[ß-gal]

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…?

Test of Positive Regulation

From Kimata et al (1997)

SQ16Is diauxic growth

apparent?

SQ17Is appearance of

ß-gal repressed by glucose?

SQ22: How to make biosensor of heavy metals

Red on MacConkey

plates