Reporter: LacZ Enzyme

Overview¶

The LacZ Enzyme Reporter is purified β-galactosidase (LacZ) added to the outer solution in a basic buffer. It converts the yellow substrate chlorophenol red-β-D-galactopyranoside (CPRG) into the purple chlorophenol red (CPR). It reads out CPRG that was released from liposomes, such as Substrate LUV: CPRG and Substrate SUV: CPRG. It is the reporter of the pH Cascade. The basic buffer raises the pH of the assay so that β-galactosidase can act.

Reference Composition¶

Enzyme Solution

Basic Buffer

Enzyme solution, a 3× solution made from the 1000 U/mL stock in the basic buffer of the next tab. Adding 30 µL of it to 60 µL of assay gives 1.2 U/mL.

Component	Stock concentration	3× concentration	Final concentration
β-galactosidase	1000 U/mL	3.6 U/mL	1.2 U/mL

Basic buffer that carries the enzyme. Its pH is 9.9, and its osmolarity is 1165 mOsm/kg, measured.

Component	Amount
Tris base, 1 M	1 mL
HCl, 37 %	2.5 µL

Expected Behavior¶

Solution¶

The LacZ Enzyme Reporter is expected to turn released CPRG from yellow to purple in solution. At 1.2 U/mL the color was visible by eye after 5.5 h at 37 °C. Data are in the Colorimetric pH Sensor, read by Colorimetric Readout.

Gels¶

The LacZ Enzyme Reporter is expected to do the same in 3.5 mg/mL low-gelling agarose, where 1.2 U/mL gave a visible color after 5.5 h at 37 °C. In 7 mg/mL low-gelling agarose, 2 U/mL gave a visible color after 2 h in all wells, including the control without pH Sensing Cells. The leakage is described on Substrate LUV: CPRG. Data are in the Colorimetric pH Sensor.