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

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

GeneticsNews

  • Seminar Monday, Oct 30John RyanImmunology, asthma

  • Printing problems


Topics for today

Topics for today

Phenotypes of strains diploid for lac

Model for positive regulation of lac operon

Experimental test of model


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

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+


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

Predict the Lac phenotype

ß-galactosidase phenotypeN no activity (or very low)

- lactose

X

- lacZ is dead

+ lactose

X

X


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

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


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

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


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

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


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

Predict the Lac phenotype

GrowthLac+ can grow on lactose

- good lacZ

- AND good lacY

lacZ+

lacY+


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

Predict the Lac phenotype

GrowthLac+ can grow on lactose

- good lacZ

- AND good lacY

X

X

Lac-cannot grow on lactose

- bad lacZ

lacZ-


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

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-


Lac phenotype strategy

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


Lac phenotype strategy1

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?


Lac phenotype

Lac Phenotype

I- P+ O+ Z+ Y+ F’( I+ P+ Oc Z- Y+)

ß-Gal phenotype?

-

lacZ repressed

(lacY constitutive)

- -

Without lactose


Lac phenotype1

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


Positive regulation of lac operon

Positive Regulation of lac operon

State

  • Plenty of glucose

Response

  • Eat it

  • Avoid other sugars

Symbol of state

  • Low cAMP

glucose

lactose

other sugars


Positive regulation of lac operon1

Positive Regulation of lac operon

State

  • No glucose

Response

  • Eat other sugars

Symbol of state

  • High cAMP

lactose

other sugars


Positive regulation

Positive Regulation

X

X

crp-

Test of modelWhat happens if...

X

X

Low expression of lac operon


Positive regulation1

Positive Regulation

X

Test of modelWhat happens if...

X

X

Low expression of lac operon

(Why defects lead to low expression?)


Positive regulation2

Positive Regulation

X

Test of modelWhat happens if...

X

X

X

Low expression of lac operon


Positive regulation3

Positive Regulation

Protein Regulation Target Acts on Influenced by

Lac repressor

CRP

Negative operator lac operon lactose

Positive

CRPbinding site

many operons

cAMP


Positive regulation sq14 superimpose camp gal on growth curve

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


Test of positive regulation sq15

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

Test of Positive Regulation

SQ16Is diauxic growth apparent?

SQ17Is appearance of ß-gal repressed by glucose?

From Kimata et al (1997)


Sq22 how to make biosensor of heavy metals

SQ22: How to make biosensor of heavy metals

Red on MacConkey plates


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