BIOL 2100 Lecture Notes - Lecture 38: Lac Operon, Lac Repressor, Replica Plating
15 Nov 2016
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Lecture 11/14
The plasmid used for our transformation in lab:
● Ori: the bidirectional origin of DNA replication
● Bla: gene for beta lactamase, an enzyme that destroys the beta-lactam ring, an
essential structure of the penicillin family of antibiotics
● GFP: gene for encoding green fluorescent protein
● araC: gene encoding a regulatory protein that interacts with arabinose and regulates
gene expression
*graph tells us that bacteria metabolize glucose before lactose
How do bacteria turn genes off and on as needed?
Replica plating:
● Grow bacteria on a master plate with complete medium (contains glucose)
● Press velvet covered block against master plate, some cells from each colony will be
transferred to the block
● Press block against replica plate containing only lactose as a nutrient, the location of
cells on the master plate and the replica plate are an exact match
● After incubation, only cells that can metabolize lactose will grow on the replica plate.
Cells that are present on the master plate but not the replica plate are unable to
metabolize lactose (mutation)
Lac Operon:
● Inducible: usually off but can be turned on, displays negative control
● Inducible enzymes function in catabolic pathways
● When lactose is added, B-galactosidase (the enzyme that cleaves lactose) increase
● Gene for B-galactosidase is called lac Z, gene for permease is called lac Y, and gene for
Transacetylase is called lac A
● Lac Z, lac Y, and lac A are all part of the lac operon and needed to metabolize lactose
● Lall is the regulatory gene outside the operon that encodes the repressor
○ The lac repressor is usually active
○ And inducer (lactose/allolactose) inactivates it
● In the absence of lactose, the repressor is active and the operon is turned off
● In the presence of lactose, the repressor is inactive and the operon is left on
● Type of lac operon mutants
○ Permease minus: lacY-
○ B-galactosidase minus: lacZ-
○ Constitutive: 3a (no repressor gene), 3b (no operator gene)
● Bacteria are naturally haploid but genes from one mutant can be put in another
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
The plasmid used for our transformation in lab: Ori: the bidirectional origin of dna replication. Bla: gene for beta lactamase, an enzyme that destroys the beta-lactam ring, an essential structure of the penicillin family of antibiotics. Gfp: gene for encoding green fluorescent protein. Arac: gene encoding a regulatory protein that interacts with arabinose and regulates gene expression. *graph tells us that bacteria metabolize glucose before lactose. Grow bacteria on a master plate with complete medium (contains glucose) Press velvet covered block against master plate, some cells from each colony will be transferred to the block. Press block against replica plate containing only lactose as a nutrient, the location of cells on the master plate and the replica plate are an exact match. After incubation, only cells that can metabolize lactose will grow on the replica plate. Cells that are present on the master plate but not the replica plate are unable to metabolize lactose (mutation)
