1. Lac Operon & Mutants

2. lac Operon lacZlacYlacAoCAPplacI 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 Acetylase allolactoseLac Repressor 38,00 kD

3. 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

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

5. What would the mutants be like? GenotypePhenotype? lacZ1?

6. lacZ Mutants Media with X-gal + IPTG Media with X-gal - IPTG Media with ONPG, toluene, + IPTG Media with ONPG, toluene, - IPTG

7. What would the mutants be like? GenotypePhenotype? lacZ1Lac - - unable to ferment lactose - white in presence of X-gal; white in presence of ONPG - also unable to convert lactose to allolactose (uninducible) lacY1?

8. lacY Mutants Media with X-gal + IPTG Media with X-gal - IPTG Media with ONPG, toluene, + IPTG Media with ONPG, toluene, - IPTG

9. What would the mutants be like? GenotypePhenotype? lacZ1Lac - - unable to ferment lactose - white in presence of X-gal; white in presence of ONPG - also unable to convert lactose to allolactose (uninducible) lacY1Lac - - 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?

10. lacO Mutants Media with X-gal + IPTG Media with X-gal - IPTG Media with ONPG, toluene, + IPTG Media with ONPG, toluene, - IPTG

11. What would the mutants be like? GenotypePhenotype? lacZ1Lac - - unable to ferment lactose - white in presence of X-gal; white in presence of ONPG - also unable to convert lactose to allolactose (uninducible) lacY1Lac - - 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 C Lac + - ferments lactose all the time – even in absence of inducer Operator that is not recognized by repressor Called constituitive lacI C Lac + - ferments lactose all the time – even in absence of inducer Repressor that doesn’t bind operator Called constituitive lacI S Lac - - unable to ferment lactose Repressor doesn’t bind allolactose Operator always blocked

12. What would the mutants be like? GenotypePhenotype? lacZ1 lacY1 lacO C lacI C lacI S

13. lacI c Mutants and lacI S Mutants Media with X-gal + IPTG Media with X-gal - IPTG Media with ONPG, toluene, + IPTG Media with ONPG, toluene, - IPTG

14. Table 4 – LacI is a trans acting factor

15. What about cAMP and CRP? AMP ------- Adenylate Cyclase ---------  cya crp CRP – cAMP Receptor Protein Or CAP – Catabolite Activator Protein

16. Let’s take a closer look

17. What would the mutants be like? GenotypePhenotype? cya- crp-

18. E. coli on minimal medium with Glycerol / IPTG Genotypeβ-galactosidase activity lac mRNA levels Wild type100 cya - <2 crp - <2

19. What effect would cAMP have? GenotypePhenotype? cya- Lac – does not ferment lactose well, even when induced crp- Lac – does not ferment lactose well, even when induced

20. E. coli on minimal medium with Glycerol / IPTG GenotypecAMP Addedβ-galactosidase activity lac mRNA levels Wild typeyes100 no100 cya - yes<2 no50 crp - yes<2 no<2

21. What would the mutants be like? GenotypePhenotype? 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