PBAD/araC-rbs-ahk4

Introduction

To establish a co-culture system, it is important that E. coli responds to signals produced by human cells. In our project, we decided to use isopentenyl adenine (iP), a kind of cytokinin, as a signal molecule. Cytokinins are the signaling molecules (or Phytohormones) that plants produce and play important roles in cell growth and differentiation. In the case of Arabidopsis thaliana, extracellular iP is received by a transmembrane receptor, AHK4. AHK4 has a histidine kinase activity, and binding of iP to AHK4 triggers auto-phosphorylation of AHK4 and the following histidine-to-aspartate phosphorelay. As a consequence, transcription from target genes is induced and/or repressed so that physiological states of plants are changed. The histidine kinase activity of AHK4 has shown to be activated depending on iP even in E. coli cells (Suzuki et al. 2001, Lukáš Spíchal et al. 2004). This fact encouraged us to use iP as a signaling molecule in our project (Read AHK4 Assay page).

Results

1. Qualitative experiment

As shown in Fig. 1, blue color was developed only when cells carried the AHK4 expressing plasmid and when the medium contained 100 microM iP. Therefore, we concluded that AHK4 could receive iP and downstream AHK4→RcsD→RscB→cps::lacZ pathway was activated as expected.

Cells were grown at room temperature on LB agar plates with and without iP. β-galactosidase activity was monitored by X-gal. Photographs were taken after 25h incubation.

2. Quantitative experiment

As shown in Fig. 2, over 1 microM of iP was required to observe the difference of β-galactosidase activity between AHK4 expressing cells and negative control cells. The β-galactosidase activity induced by 100 microM iP was 2.0-fold higher than that induced by 1 microM iP.

Cells were grown in liquid LB medium containing various concentrations of iP for overnight at 25˚C. β-galactosidase activity was monitored by the yellow color that was developed from ONPG.