Regulation of the lac Operon
An operon is a series of
adjacent genes that are coordinately regulated. This means that if one
gene is turned on, they're all turned on. Operons
are found only in prokaryotes. The lac operon consists of three genes
Lac operon schematic presentation
Glucose is E. coli's
favorite carbon source. If glucose is present, E. coli will
ignore all other sugars. E. coli needs the enzymes of the lac operon when lactose is
Repression of the operon is achieved by the action of the lac repressor, which binds to the lac operator and prevents transcription. The lac repressor is encoded by the gene lacI, which happens to be near the lac operon but is not part of it. Lactose binds to the Lac repressor and prevents it from repressing expression of the lac operon.
is an analogue of lactose that also binds to the Lac repressor, but, unlike
lactose, does not require the Lac permease to get inside of a cell. Because the molecule is
more hydrophobic than lactose
However, CAP can bind to DNA
only when cAMP is bound to CAP. So when cAMP levels in the cell are low, CAP fails to bind DNA and
thus RNA polymerase cannot begin its work, even in the absence of the
repressor. So the lac operon is under both negative
This dual system enables the
cell to make choices. What, for example, should the cell do when fed
both glucose and lactose? Presented with such a choice, E. coli
Although the presence of lactose removes the repressor, the presence of glucose lowers the level of cAMP in the cell and thus removes CAP. Without CAP, binding of RNA polymerase is inhibited even though there is no repressor to interfere with it if it could bind.
The b-Galactosidase assay
· ONPG is an analogue of lactose that is cleaved by beta-galactosidase to galactose + O-nitrophenol. ONPG is colorless, while O-nitrophenol is yellow. OD420 nm is the optical density of a solution at 420 nm, roughly the wavelength of yellow light.
· SDS and chloroform are added to the assay mixture to break open the cells. This permits the enzyme and ONPG to find each other. Fortunately, the addition of detergent and organic solvent does not affect the activity of b -galactosidase.
· Many enzymes, including b -galactosidase, prefer a reducing condition, which, in this assay, is provided by the sulfhydryl group of 2-mercaptoethanol.
· Most enzymes work better at one pH or another. b -Galactosidase likes pH 7.0
· High pH inactivates b -Galactosidase. The addition of sodium carbonate raises the pH to above pH 9.0 and immediately stops the reaction catalyzed by b -galactosidase.
Although bacteria are microscopic, they are still large
enough to scatter light. The apparent absorbance of a solution
Genotypes: Prokaryotic mutations are conventionally written as the name of the gene, usually followed by the number of the allele. A genotype containing "lacZ13" therefore indicates that the organism is altered in lacZ.
1. Always wear lab coat and gloves.
2. Treat all reagents with care so as not to contaminate them. Treat bacterial samples with care as to not contaminate the sample – or yourself!
A. Bacteria and Reagents:
E. coli strains: KL19
10 mM KCl, 1 mM MgSO4)
1 M Na2CO3 in water
B. Supplies: Pipette tips
12 sterile test tubes
C. Equipment: Pipettors
1. Preparation of work bench and supplies:
1.1 Wipe down bench with 70% EtOH
2. Assay of galactosidase activity
2.1 RE-GROW CELLS: Dilute overnight cultures
2.2 LABEL TUBES: You need twelve 7 ml test tubes, Label all of
them with some identifying mark
2.3 FILL TUBES: Fill each C tube with 2.5 ml TM. Fill each B tube with 1.5 ml Z buffer. Mark one of the A tubes as 0.
2.4 SET UP EXPERIMENTAL CONDITIONS:
The class shall work as 4 groups. Each group is in charge of one condition.
each case, after adding re-grown culture, mix contents of tube well, distribute
1.2 ml to the three remaining tubes marked A
2.5 RETAIN to SAMPLE: Place A tube marked 0 on ice, and immediately proceed to step 2.6.
2.6 BEGIN INCUBATION: Place remaining three A tubes in rack in 37° C incubator. Note the time.
2.7 DISTRIBUTE ALIQUOTS OF SAMPLE: from A tube marked 0 pipette 0.5 ml culture into a tube marked “B” and 0.5 ml culture into a tube marked “C”. Mix both well.
2.8 MEASURE CELL
DENSITY: Place C tube
2.9 PREPARE b
-GALACTOSIDASE ASSAY: To B tube
B-GALACTOSIDASE ASSAY: To the tube you placed 5 minutes ago in 27°C water bath
2.11 STOP b -GALACTOSIDASE
ASSAY: Monitor the tube in the water bath every so often. It may take few
minutes or tons of time before the color develops. When it develops a yellow
2.12 MEASURE b -GALACTOSIDASE ACTIVITY: Place B tube in spectrophotometer set up to read OD 420 nm. Note reading.
2.13 MEASURE LIGHT SCATTERING BY E.COLI: Measure OD550 of the same B tube you just measured.
2.14 SAMPLE CULTURE:
Every 30 minutes or so
3. Calculate enzyme activity:
If you had to dilute the bacteria in order to get an accurate measurement, then use:
= dilution factor x
of reaction = time of addition of sodium carbonate
1. Show in tabular form for all four strains and four different experimental conditions the ratio of induction, i.e., b-galactosidase activity at t90 divided by activity at t0. You should, of course, end up with 16 numbers.
Describe the results of these experiments against the background of how the lac operon really works
3. Suppose that your cells were not growing throughout the course of the experiment. How might that affect your results? How could you tell if your cells were indeed growing as fast as they could at all times?
4. You probably had a feeling what the results ought to be with most of the experimental conditions, but perhaps not with the glucose + IPTG condition. What do the results from this condition tell you about how the lac operon works?
Undoubtedly, not everyone's experiment gave the expected results. Now that you
are an expert in b -galactosidase assays,
speculate on what may have gone wrong
6. What experimental evidence can you provide for or against the proposition that transcription of the lac operon is feedback regulated. In other words, is the activity of the lac promoter influenced by whether the lac genes are actually being expressed?