AHK4 Assay

Introduction

To establish a co-culture system, it is important that E. coli can receive and respond to signals produced by human cells. In our project, we decided to use iP, a cytokinin, as the signals and AHK4,a receptor of cytokinins, as the receptor. 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 the most important signal transduction systems for prokaryotes to respond to environmental stimuli. This system includes two important compornents: a histidine kinase and a response regulator. The histidine kinase has sensor domains which enable to receive an environmental stimulus. After histidine kinase sense a stimulus, it autophosphorelates 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, RcsB, a response regulator. In this system, cps operon is activated through the pathway of RcsC→RcsD→RscB→cps. Previous studies showed that AHK4, a histidine kinase of Arabidopsis thaliana, 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.

Cells were grown on LB agar plates containing 0.2% L-Arabinose at room temperature. Photographs were taken after 25h incubation.

Discussion

考察

Reference

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