Team:Slovenia/PROJECT/proof/studies/betagal

Chuck Norris facts:

Introduction

Before we started working on our main project (biosynthesis), we had to make sure the zinc finger proteins actually bind to their predicted binding sites. To evaluate binding of zinc finger-enzyme fusion proteins in vitro, we used different methods and techniques, e.g  surface plasmon resonance (SPR) and electrophoretic mobility shift assay (EMSA). In addition, we designed a universal device to measure the binding of DNA binding proteins to the specific DNA sequences in vivo (scheme below).

Scheme: construction of a universal device to measure the binding of DNA binding proteins to target DNA sequence in vivo.

The universal device for testing in vivo binding of DNA binding proteins is composed of several parts (see the scheme): 1) a synthetic promoter, pSYN, into which a DNA binding sequence, specific for each tested DNA binding protein was inserted (between -35 and -10 sites of the promoter); 2) lacZ reporter gene, expression of which is controlled by pSYN; and, 3) the tested DNA binding protein under regulation of the arabinose inducible (pBAD) promoter. The principle of our device is that the binding of a DNA binding protein to its specific target sequence in pSYN promoter would result in decreased basal activity of this promoter. The activity of pSYN was monitored measuring the activity of β-galactosidase as a reporter (see scheme below). E. coli bacteria, containing our device on a low copy plasmid were grown in the presence of increasing concentrations of arabinose to induce expression of the DNA binding protein. After overnight growth in Luria Bertani broth, β-galactosidase activity was measured and results expressed in Miller Units as described in protocols.

Scheme: construction of a universal device to measure the binding of DNA binding proteins to target DNA sequence in vivo.