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rbs-ahk4

Sequence and Features

This gene is derived from Arabidopsis thaliana and encode a receptor of cytokinins, AHK4. Cytokinins are signaling molecules (Phytohormones) in plants and play important roles in cell growth and differentiation. AHK4 has a histidine kinase activity, and binding of a cytokinin 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 DNA sequences of this gene is optimized for expressing in E. coli cells considering the codon usage.

Characterization

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.

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 (which controls the production of polysaccharides) is induced through the RcsC→RcsD→RscB→cps pathway. The previous studies (Suzuki et al. 2001, Lukáš Spíchal et al. 2004) showed that AHK4 could replace RcsC 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 [http://2017.igem.org/Team:TokyoTech/Experiment/Chimeric_Transcription_Factor "Chemeric Transcription Factor"]page).

Result

The purpose of this experiment 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 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. As a quantitative experiment we cultured E. coli with various concentrations of iP in liquid medium and β-galactosidase activity was monitored by ONPG(another chromogenic substrate for β-galactosidase).

Qualitative experiment

Figure 1: Result of the qualitative experiment - 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.

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

Quantitative experiment

Figure 2: Result of quantitative experiment - Cells were grown in liquid LB medium containing various concentrations of iP for overnight at 25℃. β-galactosidase activity was monitored by the yellow color that was developed from ONPG.

As shown in Figure 2, over 1µM of iP is required for AHK4→RcsD→RscB→cps::lacZ to be activated dependent on iP concentration. The β-galactosidase activity induced by 100µM iP was 2.03-fold higher than the activity induced by 1µM iP.

Others

In our assay, BAD/araC promotor, an L-arabinose inducible promotor, was used for the expression of AHK4. Therefore, we first tried to determine appropriate L-arabinose concentration. However, during the experiments, we found following two serious problems caused by adding L-arabinose into medium.

1. Unexpectedly, high expression of β-galactosidase was observed by the addition of L-arabinose even in the absence of the AHK4 expressing plasmid; this result indicates that the native cps promoter is L-arabinose inducible.

2. Growth of the AHK4 expressing cells was severely inhibited by the addition of L-arabinose, indicating that overexpression of AHK4 is toxic to E. coli cells. Also, the ahk4 gene could not be ligated under the constitutive promoters in spite of our enormous trials.

Hence, we decided to conduct the experiments without L-arabinose. As shown above, leaky expression of AHK4 from the BAD/araC promotor in the absence of L-arabinose seemed to be enough to observe the iP-dependent expression of β-galactosidase.

Figure 3: Problems caused by 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 in the presence of L-arabinose and the growth of cells carrying AHK4 was inhibited almost completely.

Discussion

We confirmed that, in E. coli, AHK4 received extracellular iP, and as a consequence, the cps promoter was activated.

This result indicates that growth of E. coli cells can be controlled by introducing growth inhibiting factor inserted downstream of the cps promoter into the cells. As the E. coli cells grow, human cells receive more signaling molecules produced by E. coli, leading to the production of iP. Finally, growth inhibiting factor will be induced by iP and suppresses the growth rate.

In our experiment, as much as 1 µM of iP was needed to obsereve the activity of β-galactosidase as shown in result 2. However, the previous and similar studies (Lukáš Spíchal et al. 2004) showed that 0.1µM of iP was enough to trigger the response of AHK4 in E. coli. Therefore, we may have to consider amplifying the output signal of the pathway. For example, such amplification can be achieved by inserting the "cps promotor-gene of interest" gene cassette into a high-copy plasmid or by combining the cps promoter with the strong “T7 RNA polymerase–T7 promoter” system (Nakashima et al. 2014).

As another improvement, it is possible to slightly increase the expression of AHK4 by using promoter which is leakier than the BAD/araC promoter, such as lac promoter.

Material and Method

Plasmids

• Sample

Ptet – rbs – ahk4 (pSB1C3)

• Negative control

pSB1C3

Construction

• Strain

All the plasmids were prepared in E. coli KMI002 strain.

Qualitative experiment

1.- LB agar plates containing chloramphenicol (34 µg/mL) were prepared.

2.- 50 µl of X-Gal (50 mg/ml), 10 µl of 100 mM iP or DMSO as a control, and 40 µl of LB medium was mixed in microtubes. Then the solutions were applied to the agar plates.

3.- Samples were inoculated and incubated at room temperature.

4.- Photographs were taken after sufficient blue color was developed.

Quantitative experiment

1.- Overnight culture of samples were prepared in 2 ml of LB medium containing chloramphenicol (34 µg/mL) at 25℃.

2.- Samples were diluted for 2000-fold in 1ml of fresh LB medium containing chloramphenicol (34 µg/mL) and various concentration of IP (10 nM-100 µM). Cells were also inoculated into medium containing DMSO instead of iP.

3.- Samples were cultured overnight at 900 rpm at 25℃.

4.- Cells were collected by centrifugation at 10,000 × g for 10min.

5.- All of supernatant was discarded and then cells were resuspended in 500 µL of PBS buffer containing 1 mM MgSO4 and 1 mM dithiothreitol (DTT). Also 500 µL of the same buffer in was prepared as a control for spontaneously splitting of ONPG.

6.- 20 µL of each suspension was added into 180µL of the buffer used above and Abs600 was measured and recorded by a microplate reader.

7.- 10µL of 0.1% SDS and 10 µL of chloroform was added into each tube including the control and vortexed for 15sec.

8.- Tubes were heated at 28℃ for 5min.

9.- 100 µL of ONPG (4 mg/mL) was added to each tube and incubated at 37℃ for 30min. ONPG was dissolved in the buffer used above.

10.- After 30min incubation, tubes were heated at 65℃ for 10min to inactivate β-galactosidase.

11.- All samples were centrifuged at 15,000 rpm for 10min.

12.- Abs420 of supernatant was measured and recorded by a microplate reader. The control was used as a blank.

13.- Relative β-galactosidase activity was calculated by following formula:

Relative β-galactosidase activity = Abs420 [-] / (Abs600 [-]×10×30 [min])

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.

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.

Nakashima, N., Akita, H. and Hoshino, T. (2014) Establishment of a novel gene expression method, BICES (biomass-inducible chromosome-based expression system), and its application to the production of 2,3-butanediol and acetoin. Metab Eng. 25 :204-214.

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.

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.