B-Galactosidase Assay (A better Miller)

β-Galactosidase Assay

Background

β-Galactosidase is encoded by the lacZ gene of the lac operon in E. coli. It is a large (120 kDa, >1000 amino acids) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentratration.

So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the lacZ gene and use beta-Gal levels as a readout for promoter activity under various conditions. In 1974, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".

1 Miller Unit = 1000 * (Abs420 - (1.75*Abs550))/(t * v * Abs600)

where: Abs420 is the absorbance of the yellow o-nitrophenol, Abs550 is the scatter from cell debris, which, when multiplied by 1.75 approximates the scatter observed at 420nm, t = reaction time in minutes v = volume of culture assayed in milliliters Abs600* reflects cell density.

• Note that this value is different for each spectrophotometer used and should be calibrated by plating dilutions of known Abs600 cultures to determine the colony-forming units per Abs600.

In his book, Dr. Miller explains that this formula yields approximately 1 Miller Unit for uninduced E. coli (low β-Gal production) and approximately 1000 units for a fully induced culture (grown on lactose or IPTG).

In my experience, cultures of MG1655 induced with 1 mM IPTG in log phase have 1500-1800 Miller units. The reason for the difference is not known, but I suspect it stems from differences in the Abs600/cell density between Dr. Miller's spectrophotometer and the one I use and the fact I do my Miller assays at 30 degC (for convenience) whereas Dr. Miller performed his assays at 28 degC. I have made promoter fusions that generate ~40,000 Miller units; however, as will be discussed below, this is too high for the assay and so the protocol was changed to lower this value.

Protocol

The protocol I use is derived from a paper by Zhang and Bremer (JBC 270, 1995) in which the original Miller protocol was greatly simplified to allow more samples to be measured with less manipulation.

In short, the protocol consists of measuring the cell density of a culture of bacteria (Abs600), then removing an aliquot of the cells from the cuvette and mixing them with a "permeabilization" solution that contains detergent which disrupts the cell membranes (but leaves the beta-Gal intact). This kills the cells and stops translation. After incubation, an ONPG "substrate" solution is added and the yellow color allowed to develop. A "stop" solution is then added and the absorbance of o-nitrophenol is measured.

• Grow cultures under whatever conditions you wish to test.

• During growth, pre-measure 80 uL aliquots of permeabilization solution into 1.5 mL microfuge tubes and close them.

• measure Abs600 and RECORD IT!

• Remove a 20 uL aliquot of the culture and add it to the 80 uL of permeabilization solution.

The sample is now stable for several hours. This allows you to perform time-course experiments.

• After the last sample is taken, move the samples and the Substrate solution to the 30 degree warm room for 20-30 minutes.

• Add 600 uL of Substrate solution to each tube and NOTE THE TIME OF ADDITION.

• After sufficient color has developed, add 700 uL of Stop solution, mix well, and NOTE THE STOP TIME.

• After stopping the last sample (some may take longer than others, but generally they are done in 30-90 minutes), transfer the tubes to a microfuge and spin for 5-10 minutes at full speed.

• Carefully remove the tubes from the centrifuge and transfer solution from the TOP of the tubes to your cuvette(s). You are trying to avoid having particulate matertial in the cuvette so that scattering will not influence the reading.

• Record Abs420. This should be less than 1 and greater than 0.05. If it's a bit outside of this range, don't sweat it.

Calculate Miller Units as:

1000 * (Abs420)/((Abs600 of culture sampled)*(volume [0.02 mL])*(reaction time))

Recipes

Permeabilization Sulution

You need 80 uL per sample.

100 mM dibasic sodium phosphat(Na2)

(the Zhang protocol has 200 mM sodium phosphate. I could never get this into solution with the other components, no matter what I tried so I backed it off to 100 mM. I have even used 50 mM with no detectable change)

20 mM KCl

2 mM MgSO4

0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide)

0.4 mg/mL sodium deoxycholate

5.4 uL/mL beta-mercaptoethanol

Substrate solution

60 mM NaHPO4

40 mM NaH2PO4

1 mg/mL o-nitrophenyl beta-D-Galactoside (ONPG)

2.7 uL/mL beta-mercaptoethanol

(The Zhang protocol also has 20 ug/mL CTAB and 10 ug/mL deoxycholate, I leave these out figuring that there is still plenty from the permeabilization solution and, if they aint't dead yet, they win't gonna be)

Stop solution

1 M NaCO3

The high pH of the stop solution denatures the beta-Gal and approzimately doubles the yellow color of the reaction.

so much for now