LIFESCI 23L Lecture Notes - Lecture 3: Lac Operon, Lac Repressor, Lactose Intolerance
Lecture C: b-galactosidase Assay
OVERVIEW OF SKILLS/CONCEPTS
● Working w/ E. Coli cell cultures, using enzyme activity assay, and use of spectrophotometer
● Cocnepts: lac operon and function; protein activity assay, spectophotometer (cell density)
● Skills: pipetting, spectrophotometer, handling/disposal of bacterial cells
BACKGROUND
● LACTASE: enzyme that breaks down dissacharide lactose (galactose+glucose)
○ Beta-galactosidease → breaks beta 1, 4 glycosidic bonds
○ Sugar found in milk; all mammals express lactase at birth, but many stop producing lactase
(lactose intolerant)
■ Lactose = found in milk and dary product
■ Ability to continue drinking milk as an adult=mutation
● Mutation didn’t spread until humans started using dairy in diet ~8000 years ago
○ Lactase and its regulation extensively studied in e. Coli, a common bacteria used in labs
■ Uses genes + lac operon to metabolize lactose
● Gene expression
○ DNA → RNA → protein → carrying out functions in cell
○ Trasncription: DNA → RNA
■ Promotor before gene; marks start site of transcription
○ Translation: RNA → protein
■ mRNA is recognized by ribosome
○ Activity of a specific enzyme can be regulated by inhibiting the protein directly or by regulating
its transcription/translation
■ Lactase is operated @ transcription level
● LAC OPERON: mechanism for e. Coli to detect lactose levels and respond to them
○ Lac operon includes
■ Three genes: lac Z, lac Y, lac A → structural genes → codes for PROTEINS
● Lac Z → lactase found in cytosol
○ Lactase has 2 functions
■ Breaks lactose into glucose and galactose for energy
■ Isomerize lactose into allolactose, an indicator of the presence of
lactose in the cell
● Lac Y → channel protease called permease, found in plasma membrane
○ Allows lactose from environment into bacteria through plasma membrane
(sugar=polar molecules)
● Lac A → encodes minor enzyme that chemically modifies lactose during its
metabolsim, also found in cytoplasm
○ Not very important, will not go into detail
■ Regulatory elemetns that controls expression
● Common promotor: lac P
● Operator: lac O → binding site for lac repressor
○ Lac repressor (DNA binding protein) encoded by gene lac I (not part of
lac operon)
■ Lac repressor gene product found in cytosol
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Document Summary
Working w/ e. coli cell cultures, using enzyme activity assay, and use of spectrophotometer. Cocnepts: lac operon and function; protein activity assay, spectophotometer (cell density) Skills: pipetting, spectrophotometer, handling/disposal of bacterial cells. Lactase: enzyme that breaks down dissacharide lactose (galactose+glucose) Beta-galactosidease breaks beta 1, 4 glycosidic bonds. Sugar found in milk; all mammals express lactase at birth, but many stop producing lactase (lactose intolerant) Lactose = found in milk and dary product. Ability to continue drinking milk as an adult=mutation. Mutation didn"t spread until humans started using dairy in diet ~8000 years ago. Lactase and its regulation extensively studied in e. coli, a common bacteria used in labs. Uses genes + lac operon to metabolize lactose. Dna rna protein carrying out functions in cell. Promotor before gene; marks start site of transcription. Activity of a specific enzyme can be regulated by inhibiting the protein directly or by regulating its transcription/translation.
