Regulation of the lac Operon

I. Principle

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 (lacZ, lacY, and lacA) preceded by a promoter “P” and an operator “0”.  LacZ encodes galactosidase, an enzyme that catalyzes the breakdown of lactose (milk sugar) to glucose + galactose.  LacY encodes lactose permease, a protein that sits in the cell membrane and facilitates the entry of lactose into the cell.  LacA encodes acetyl transferase that transfers acetyl groups from acetyl-CoA to the 6-hydroxyl of certain galactopyranosides.  These genes are involved with lactose metabolism.  Therefore it is reasonable that they should be turned on or off according to the same nutritional conditions.

II. Explanation
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 present (e.g., when E. coli is swimming in a gut full of milk) but there isn't anything easier to eat around, like glucose.  When lactose is not present, there is no need for the lac operon to be expressed.

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.

IPTG 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 (owing to the isopropyl substitute), it can swim through the membrane directly. Absence of the lac repressor is essential but not sufficient for effective transcription of the lac operon. The activity of RNA polymerase also depends on the presence of another DNA-binding protein called catabolite activator protein or CAP. Like the lac repressor, CAP has two types of binding sites: One binds the nucleotide cyclic AMP; the other binds a sequence of 16 base pairs upstream of the promoter.

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 (the repressor) and positive (CAP) control.  Why?

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 (for reasons about which we can only speculate) chooses glucose. It makes its choice by using the interplay between these two control devices.

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 (measured as its optical density -- OD) is proportional to the number of bacteria in the solution, so long as the solution isn't too dense. The wavelength at which light scattering is measured doesn't matter much, so long as the wavelength is distant from 420 nm, the wavelength at which O-nitrophenol absorbs.

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.

III. General Safety Requirements

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!

IV. Essentials

A. Bacteria and Reagents:

E. coli strains: KL19 (wild-type)

Media: LB: (per liter) 10 g tryptone, 5 g yeast extract, 10 g NaCl

Reagents: IPTG (100 mM isopropyl thiogalactoside, in water)

Lactose (8% in water, filter sterilized)
glucose (20% in water, filter sterilized)
ONPG (4g/ml ortho-nitrophenol galactoside, in phosphate buffer)
Phosphate buffer (0.1 M phosphate, pH 7.0, autoclaved)
Z buffer (50 mM 8-mercaptoethanol, 100 mM NaPhosphate (pH 7.0),

              10 mM KCl, 1 mM MgSO4)
1 M Na2CO3 in water
SDS (20% stock solution of sodium dodecyl sulfate)
chloroform

B. Supplies:  Pipette tips

                     Pipettes

                     12 sterile test tubes

                     Ice buckets

C. Equipment: Pipettors

    Water baths

                        Spectrophotometer

Cuvettes

V. Protocol

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 (KL19) 1:25 in fresh growth medium, pre-warmed to 37° C. Put diluted cultures at 37° C for 30-60 min.

       2.2 LABEL TUBES: You need twelve 7 ml test tubes, Label all of them with some identifying mark (e.g. your initials). Label four of them C (for cell density), four of them B (for b -galactosidase assay) and four of them as A.

       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.

(Grp1) No addition: Add 1.2ml re-grown culture to each tube A.

(Grp2) +IPTG: Add 4.8ml re-grown culture to the A tube marked 0, to which has been previously added 15µl 100mM IPTG.

(Grp3) +lactose: Add 4.8ml re-grown culture to the A tube marked 0, to which has been previously added 375 µl 8% lactose.

(Grp4) + glucose, + IPTG: Add 4.8 ml re-grown culture to the A tube marked 0, to which has been previously added 150µl 20% glucose and 15ul IPTG.

In each case, after adding re-grown culture, mix contents of tube well, distribute 1.2 ml to the three remaining tubes marked A (leaving 1.2 ml in the A tube marked 0), and proceed immediately to step 2.5.

2.5  RETAIN t_o 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 (to which you just added culture) in spectrophotometer set up to read OD600. Note reading. (NOTE:  ALL SPECS SHOULD BE BLANKED WITH WATER.)

2.9  PREPARE b -GALACTOSIDASE ASSAY: To B tube (to which you just added culture) add 50µl of 20% SDS and 50µl chloroform. Vortex for 10 seconds. Place tube in rack in 27°C water bath for about 5 minutes.

2.10  BEGIN B-GALACTOSIDASE ASSAY: To the tube you placed 5 minutes ago in 27°C water bath (tube B), add 400µl ONPG (b -galactosidase substrate). Briefly mix and return to water bath. Note the time.

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 color (light but unambiguous), stop the reaction by adding 1ml sodium carbonate. Note the time. Mix well. Once sodium carbonate has been added, the tube can remain at your bench until a spectrophotometer is free. If the color develops within several seconds, that's too fast to be accurately measured. In that case, dilute the cells by a factor of five in growth medium in a new tube and begin the assay from the beginning.

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 (i.e., at t₃₀, t₆₀, and t₉₀) take one of the A tubes out of the incubator and place it on ice or immediately repeat steps 7-12. Note the time.

3. Calculate enzyme activity: (use the following formula)

Activity = (OD₄₂₀ - OD₅₅₀ / (OD₆₀₀ x duration of reaction)

If you had to dilute the bacteria in order to get an accurate measurement, then use:

Activity = dilution factor x (OD₄₂₀ - OD₅₅₀ / (OD₆₀₀ x duration of reaction)

[Duration of reaction = time of addition of sodium carbonate (stop) minus time of addition of ONPG (start)]

Questions:

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.

2. Describe the results of these experiments against the background of how the lac operon really works (your conception of which should be outlined in the Introduction to your report).

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?

5. 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 (if, indeed, anything did).

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?