Lac Operon Mutants lac Operon lac I Lac

Lac Operon & Mutants

lac Operon lac. I Lac Repressor 38, 00 k. D CAP p o lac. Z allolactose lac. Y lac. A Acetylase Lac Permease, (symporter) 1. 5 kb m. RNA, 46, 500 k. D β-galactosidase, 3. 1 kb m. RNA, 120 k. D (1) glc-gal glc + gal (2) β-1 -4 lactose β-1 -4 allolactose

Wild type Media 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 type Media with ONPG, toluene, + IPTG Media with ONPG, toluene, - IPTG *X-gal and IPTG move freely into cells because of toluene

What would the mutants be like? Genotype Phenotype? lac. Z 1?

Media with X-gal + IPTG lac. Z Mutants Media with ONPG, toluene, + IPTG Media with X-gal - IPTG Media with ONPG, toluene, - IPTG

What would the mutants be like? Genotype Phenotype? lac. Z 1 lac. Y 1? Lac- - unable to ferment lactose - white in presence of X-gal; white in presence of ONPG - also unable to convert lactose to allolactose (uninducible)

Media with X-gal + IPTG lac. Y Mutants Media with ONPG, toluene, + IPTG Media with X-gal - IPTG Media with ONPG, toluene, - IPTG

What would the mutants be like? Genotype Phenotype? lac. Z 1 Lac- - unable to ferment lactose - white in presence of X-gal; white in presence of ONPG - also unable to convert lactose to allolactose (uninducible) lac. Y 1 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 lac. O?

lac. O Mutants Media with X-gal + IPTG Media with ONPG, toluene, + IPTG Media with X-gal - IPTG Media with ONPG, toluene, - IPTG

What would the mutants be like? Genotype Phenotype? lac. Z 1 Lac- - unable to ferment lactose - white in presence of X-gal; white in presence of ONPG - also unable to convert lactose to allolactose (uninducible) lac. Y 1 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 lac. OC Lac+ - ferments lactose all the time – even in absence of inducer Operator that is not recognized by repressor Called constituitive lac. IC Lac+ - ferments lactose all the time – even in absence of inducer Repressor that doesn’t bind operator Called constituitive lac. IS Lac- - unable to ferment lactose Repressor doesn’t bind allolactose Operator always blocked

What would the mutants be like? Genotype Phenotype? lac. Z 1 lac. Y 1 lac. OC lac. IS

lac. Ic Mutants and lac. IS Mutants Media with X-gal + IPTG Media with ONPG, toluene, + IPTG Media with X-gal - IPTG Media with ONPG, toluene, - IPTG

Table 4 – Lac. I is a trans acting factor

What about c. AMP and CRP? cya AMP ------- Adenylate Cyclase ----- crp CRP – c. AMP 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 m. RNA levels Wild type 100 cya- <2 <2 crp- <2 <2

What effect would c. AMP 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 c. AMP Added β-galactosidase activity lac m. RNA levels Wild type yes 100 no 100 yes <2 <2 no 50 50 yes <2 <2 no <2 <2 cya- crp-

What would the mutants be like? Genotype Phenotype? cya- crp- Lac – does not ferment lactose well, even when induced Adenylate Cyclase does not make c. AMP Not activated Lac – does not ferment lactose well, even when induced Adenylate Cyclase does not make CRP Not activated