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pBad/araC-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 ^[1]. As a consequence, transcription from target genes is induced and/or repressed so that physiological states of plants are changed. Since overexpression of AHK4 seemes to be toxic for E. coli, the expression is tightly regulated by BAD/araC promoter, an L-arabinose inducible promoter.

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 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, 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 of Arabidosis 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.

Result

In our assay, we used bad/araC promotor, a L-arabinose inducible promotor, for the expression of AHK4. Therefore, we fist try to determine appropriate L-arabinose concentration. But through the experiment, we found two big problem caused by L-arabinose in medium. Firstly, cps promotor was activated by different pathway from AHK4→RcsD→RscB→cps::lacZ pathway under the existence of L-arabinose as shown in Fig and Fig. Secondly, the growth of AHK4 carrying cells were inhibited when L-arabinose exists in medium. Considering these two facts, we decided to conduct assays without L-arabinose.

[Fig.1 Result of the qualitative experiment]

As shown in Fig, blue color was developed only when cells were carrying AHK4 and when the medium was containing iP.

As shown in fig , 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.

Discussion

Through 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.

Material and Method

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.

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

[1] The Arabidopsis sensor His-kinase, AHk4, can respond to cytokinins, 2001. Suzuki T. et.al