Assay for ß-galactosidase in E. Coli

 

 

 

 

 

 

1. Constitutive - those enzymes that are continuously synthesized in the cell (e.g. the enzymes of glycolysis or the lac repressor protein);

 

2 Inducible - those enzymes that are synthesized in the cell only when an inducer (signal) is present in the cell (e.g. ß-galactosidase or the enzymes in the tryptophan biosynthesis pathway).

 

This procedure is designed to induce and measure the level of ß-galactosidase in E. coli. Cells grown in the absence of lactose do not synthesize ß-galactosidase. If these cells are placed in a medium containing lactose, ß-galactosidase is produced within minutes, enabling the cells to use lactose as a source of carbon and energy for growth.

Some compounds are inducers of the synthesis of ß-galactosidase and some compounds are substrates for the activity of ß-galactosidase. This week you will investigate the ability of four compounds to induce the activity of ß-galactosidase. The four compounds are: lactose, isopropyl-ß-thiogalactoside (IPTG), phenyl-ß-galactoside (PBG) and glucose (GLU).

 

We will use a non-biological (artificial) substrate for the enzyme, Ortho-nitrophenyl-ß-galactoside (ONPG). In the presence of ß-galactosidase ONPG is converted to galactose and Ortho-nitrophenyl (ONP). E. coli cells contain no enzymes capable of degrading ONP further.

ONPG is colorless. ONP is also colorless at neutral or acid pH, but in an alkaline solution it is bright yellow. The amount of yellow color can be measured in a spectrophotometer and can be used as a measure of the amount of ONP formed in a given time. Since ONP is a product of ß-galactosidase activity, the spectrophotometric measurements may be used as an assay for the enzyme.

 

a) 4 ml of starved E. coli cells (1 x 107 cells/ml).

b) 0.2 ml of .002 M inducer (LAC, GLU, IPTG, PBG, or dH2O)

Put a cap on each tube, place in a 37 C water bath and aerate (shake) for 30 minutes.

 

 

1) One drop sodium desoxycholate (1.0 mg/ml)
2) One drop toluene

Cap, place in a 37 o C water bath and aerate or shake for 10 minutes. This preparation may be used for enzyme assays. Keep it in an ice bucket and only remove sample when needed for assays.

 

1) 2.0 ml of 0.1 M sodium phosphate buffer (pH 7)
2) 2.0 ml lysed E. coli preparation
3) 0.2 ml of 0.01 M ONPG (Substrate)
Incubate for 15 minutes at 37 C without shaking. Stop the reaction by adding 0.5 ml 2M sodium carbonate. This will make the solution alkaline (pH>8) and denature the enzyme. Read the absorbance at 420nm in a spectrophotometer. If the compound is an inducer, more enzyme will be formed, more substrate (ONPG) will be converted to a yellow product and the absorbance will be higher.