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iGEM Tokyo Tech

AHK4 Assay

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 (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 i in E. coli cells (Suzuki et al. 2001). This fact encouraged us to use iP as a signale molecule in our project..

A His-to-Asp phosphorelay system is one of the 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 a sensor domain which receives an environmental stimulus. After the histidine kinase sense a stimulus, autophosphorylation takes place 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 in E. coli is composed of three components: RcsC, a histidine kinase, RcsD, a histidine-containing phosphotransmitter, and RcsB, a response regulator. This system is activated after stress exposure such as osmolality shock; cps operon promoter is promoter (which controls the production of polysaccharides) isinduced through the RcsC→RcsD→RscB→cps pathway. The previous study (Suzuki et al. 2001) showed that AHK4, could replace in E. coli and cps operon expression was induced depending on iP addition.

Since iP and AHK4 are only used in plants in nature, 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.Fortunately, heterologous synthesis of iP in human cells seemed to be easy for us, because introduction of only two A. thaliana genes to human cells was sufficient to do so (see the nnn page).

Summary

The purpose of experiments on this page is to confirm that AHK4 protein expressed in E. coli can receive iP produced by human cells, and the fllowing AHK4→RcsD→RscB→cps pathway is activated. To this end, we chose the E. coli KMI002 strain as a host to express AHK4. The KMI002 strain habors cps::lacZ fusion gene and the activation of AHK4→RcsD→RscB→cps::lacZ pathway can be confirmed with the activity of β-galactosidase.

As a qualitative experiment, we observed it if AKH4 expressing cells develop blue color in the presence of both iP and X-gal (a chromogenic substrate forβ-galactosidase) on agar plates.

As a quantitative experiment, we cultured the E. coli cells with various concentrations of iP in liquid medium and measured β-galactosidase activity using ONPG (another chromogenic substrate forβ-galactosidase).

Results

1. Qualitative experiment

As shown in Fig1, blue color was developed only when cells were carried the AHK4 expressing plasmid and when the medium was contained 100 µM iP. Therefore, we concluded that AHK4 could receive iP and downstream AHK4→RcsD→RscB→cps::lacZ pathway was activated asexpected.

Fig1. Result of the qualitative experiment

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.

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 adding L-Arabinose in medium.

1. cps promoter was induced by different pathway from 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.

Discussion

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

However, we need as much as 1µM of iP to see a activity of β-galactosidase as shown in result 2. But other study (Lukáš Spíchal　et al. 2004) 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 leakier than bad/araC promoter, such as lac promoter.

Reference

Suzuki, T., Miwa, K., Ishikawa, K., Yamada, H., Aiba, H. and Mizuno, T. (2001) The Arabidopsis Sensor His-kinase, AHK4, Can Respond to Cytokinins. Plant Cell Physiol. 42: 107-113.

Yamada, H., Suzuki, T., Terada, K., Takei, K., Ishikawa, K., Miwa, K., Yamashino, T. and Mizuno, T. (2001) The Arabidopsis AHK4 Histidine Kinases is a Cytokinin-Binding Receptor that Transduces Cytokinin Signals Across the Membrane. Plant Cell Physiol. 42: 1017-1023.

Spíchal, L., Rakova, N.Y., Riefler, M., Mizuno, T., Romanov, G.A.,Strnad, M. and Schmülling, T. (2004) Two Cytokinin Receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, Differ in their Ligand Specifity in a Bacterial Assay. Plant Cell Physiol. 45: 1299-1305.

Klimeš, P., Turek, D., Mazura, P., Gallová, L., Spíchal, L. and Brzobohatý, B. (2017) High Throughput Screening Method for Identifying Potential Agonists and Antagonists of Arabidopsis thaliana Cytokinin Receptor CRE1/AHK4. Frontiers in Plant Science.

Mizuno, T. and Yamashino, T. (2010) BIOCHEMICAL CHARACTERIZATION OF PLANT HORMONE CYTOKININ-RECEPTOR HISTIDINE KINASES USING MICROORGANISMS. Methods in Enzymology: 335-344.

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      	<figcaption style="font-family: Poppins;font-size: 16px">Fig1. Result of the qualitative experiment</figcaption>
          <p style="font-family: Poppins;font-size: 10px"><p style="text-indent:1em"> 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.