Lac Operon & Mutants

lac Operon

lacZ lacY lacAoCAP placI

β-galactosidase, 3.1 kb mRNA, 120 kD(1) glc-gal glc + gal(2) β-1-4 lactose β-1-4 allolactose

Lac Permease, (symporter)1.5 kb mRNA, 46,500 kD

AcetylaseallolactoseLac Repressor

38,00 kD

Wild typeMedia with X-gal + IPTG Media with X-gal - IPTG

*Uptake of X-gal and IPTG controlled by Lac Permease*Small amount of Lac Permease present in membrane at all times*IPTG not degraded

Wild typeMedia with ONPG, toluene,

+ IPTGMedia with ONPG, toluene,

- IPTG

*X-gal and IPTG move freely into cells because of toluene

What would the mutants be like?Genotype Phenotype?

lacZ1?

lacZ MutantsMedia with X-gal + IPTG Media with X-gal - IPTG

Media with ONPG, toluene, + IPTG

Media with ONPG, toluene, - IPTG

What would the mutants be like?Genotype Phenotype?

lacZ1 Lac- - unable to ferment lactose- white in presence of X-gal; white in presence of ONPG- also unable to convert lactose to allolactose (uninducible)

lacY1?

lacY MutantsMedia with X-gal + IPTG Media with X-gal - IPTG

Media with ONPG, toluene, + IPTG

Media with ONPG, toluene, - IPTG

What would the mutants be like?Genotype Phenotype?

lacZ1 Lac- - unable to ferment lactose- white in presence of X-gal; white in presence of ONPG- also unable to convert lactose to allolactose (uninducible)

lacY1 Lac- - unable to ferment lactose in whole cells-unable to take up lactose, and so unable to convert lactose to allolactose (uninducible)- Wild type in lysed cells – called “cryptic” mutants

lacO?

lacO MutantsMedia with X-gal + IPTG Media with X-gal - IPTG

Media with ONPG, toluene, + IPTG

Media with ONPG, toluene, - IPTG

What would the mutants be like?Genotype Phenotype?

lacZ1 Lac- - unable to ferment lactose- white in presence of X-gal; white in presence of ONPG- also unable to convert lactose to allolactose (uninducible)

lacY1 Lac- - unable to ferment lactose in whole cells-unable to take up lactose, and so unable to convert lactose to allolactose (uninducible)- Wild type in lysed cells – called “cryptic” mutants

lacOC Lac+ - ferments lactose all the time – even in absence of inducerOperator that is not recognized by repressorCalled constituitive

lacIC Lac+ - ferments lactose all the time – even in absence of inducerRepressor that doesn’t bind operatorCalled constituitive

lacIS Lac- - unable to ferment lactoseRepressor doesn’t bind allolactoseOperator always blocked

What would the mutants be like?Genotype Phenotype?

lacZ1

lacY1

lacOC

lacIC

lacIS

lacIc Mutants and lacIS Mutants Media with X-gal + IPTG

Media with X-gal - IPTG

Media with ONPG, toluene, + IPTG

Media with ONPG, toluene, - IPTG

Table 4 – LacI is a trans acting factor

What about cAMP and CRP?

AMP ------- Adenylate Cyclase ---------

cya

crpCRP – cAMP Receptor Protein Or CAP – Catabolite Activator Protein

Let’s take a closer look

What would the mutants be like?Genotype Phenotype?

cya-

crp-

E. coli on minimal medium with Glycerol / IPTG

Genotype β-galactosidase activity

lac mRNA levels

Wild type 100 100

cya- <2 <2

crp- <2 <2

What effect would cAMP have?Genotype Phenotype?

cya- Lac – does not ferment lactose well, even when induced

crp- Lac – does not ferment lactose well, even when induced

E. coli on minimal medium with Glycerol / IPTG

Genotype cAMP Added β-galactosidase activity

lac mRNA levels

Wild type yes 100 100no 100 100

cya- yes <2 <2no 50 50

crp- yes <2 <2no <2 <2

What would the mutants be like?Genotype Phenotype?

cya- Lac – does not ferment lactose well, even when induced

Adenylate Cyclase does not make cAMP

Not activated

crp- Lac – does not ferment lactose well, even when induced

Adenylate Cyclase does not make CRP

Not activated