AHK4 Assay

Introduction

To establish a co-culture system, it is important that E. coli can response to signals produced by human cells. In our project, we decided to use isopentenyl adenine (iP), a cytokinin, as the signals and AHK4, a receptor of cytokinins exists in Arabidopsis thaliana, as the receptor. This AHK4 can respond to iP by using a Histidine-to-Aspartate phosphorelay system existing in E. coli.

A histidine-to-aspartate phosphorelay system is one of most important signal transduction systems for prokaryotes to respond to environmental stimuli. This system includes two important components: a histidine kinase and a response regulator. The histidine kinase has sensor domains which enable to receive an environmental stimulus. After a histidine kinase sense a stimulus, it autophosphorylates and then the phosphate group is transferred to the response regulator, which in turn, promote expression of a certain gene corresponding to the stimulus.

One of the His-to-Asp phosphorelay systems used in E. coli is composed of three components: RcsC, a histidine kinase, RcsD, a histidine-containing phosphotransmitter, and RcsB, a response regulator. In this system, cps operon is activated through the pathway of RcsC→RcsD→RscB→cps. Previous studies show that AHK4, a histidine kinase, can also take advantage of RcsD→RscB→cps pathway in E. coli by receiving cytokinins.

Since iP and AHK4 are only used in plants, we considered that employing this AHK4→RcsD→RscB→cps pathway enable us to establish communication between human cells and bacteria without activating any other unexpected genes.

Summary

The purpose of experiments on this page is to confirm that AHK4 can receive iP, a signal molecule produced by human cells, and AHK4→RcsD→RscB→cps pathyway will be activated in turn. To see the activation of the pathway we used KMI002 strain as a carrier of AHK4. This KMI002 possesses cps::lacZ fusion gene and the activation of AHK4→RcsD→RscB→cps::lacZ pathway can be observed through the activity of β-galactosidase.

As a qualitative experiment, we monitored if AKH4 carrying KMI002 develops blue color under the existence of iP and X-gal on agar plates. Therefore we concluded that AHK4 could receive iP and downstream pathway was activated.

As a quantitative experiment, we cultured E. coli with various concentrations of iP in liquid medium and measured β-galactosidase activity by using ONPG.

Results

1. Qualitative experiment

As shown in Fig1, blue color was developed only when cells were carrying AHK4 and when the medium was containing iP. Therefore, we concluded that AHK4 could receive iP and downstream AHK4→RcsD→RscB→cps::lacZ pathway was activated.

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 Fig2, over 1µM of iP is required to see a difference of β-galactosidase activity between AHK4 carrying cells and negative control cells. The β-galactosidase activity induced by 100µM iP was 2.03-fold higher than the activity induced by 1µM iP.

Cells were grown in liquid LB medium containing various concentrations of iP for overnight at 25℃ at 900 rpm. β-galactosidase activity was monitored by ONPG.

3. Others

In our assay, we used bad/araC promotor, a L-Arabinose inducible promotor, for the expression of AHK4. Therefore, we fist tried to determine appropriate L-Arabinose concentration. But through the experiment, we found following two big problems caused by L-Arabinose in medium.

1. cps promoter was induced by different pathway than AHK4→RcsD→RscB→cps::lacZ pathway under the exsistence of L-Arabinose.

2. The growth of AHK4 carrying cells were inhibited by actively expressing AHK4 receptor by L-Arabinose.

Hence, we decided to conduct the experiments without L-Arabinose.

Cells were grown on LB agar plates containing 0.2% L-Arabinose with and without iP at room temperature. Photographs were taken after 25h incubation. Negative control cells developed blue color under the existence of L-Arabinose and compared with negative control cells, growth of AHK4 carrying cells were inhibited.

Discussion

Through the experiments we could confirm that AHK4 can receive iP and cps promotor will be activated. This result showed us we can control the growth of bacteria by fusing a gene of growth inhibiting factor, such as mazF, downstream of the promotor.

However, we need as much as 1µM iP to see a activity of β-galactosidase. But other study showed that 0.1µM of iP can trigger the response of AHK4. Therefore, we consider that we can amplify the output of the pathway by inserting cps promotor and downstream gene into a high-copy plasmid.

For another improvement, we consider that we can slightly increase the expression of AHK4 by using promoter which is laekier than bad/araC promoter.

Reference

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