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(tra box)7-CMVmin-atipt4-IVS-IRES-log1-polyA

Sequence and Features

tra box : Mammalian Tra-R inducible element BBa_K553021

These two genes(apipt4 and log1) are derived from Arabidopsis thaliana and encode enzymes necessary for synthesizing iP (isopentenyladenine) in mammalian cells. iP is a kind of cytokinins that are signaling molecules (Phytohormones) in plants and play important roles in cell growth and differentiation. When these genes are introduced to human cells, EA.hy926, the cells produce iP heterologously. AtIPT4 has the adenylate dimethylallyltransferase ([http://www.genome.jp/dbget-bin/www_bget?ec:2.5.1.112 [EC:2.5.1.112] ]: cytokinin synthase) activity and catalyzes the transfer of an isopentenyl group from dimethylallyl diphosphate (DMAPP) to ATP and ADP, producing cytokinin nucleotides ^[1]. Note that cytokinin nucleotides are the immature form. LOG1 has the phosphoribohydrolase activity and converts inactive cytokinin nucleotides to the biologically active free-base forms ^[2].

The expression of these genes are induced by a chemeric transcription factor which consist of eukaryotic activation domain of NF-κB p65 and prokaryotic transcription factor, TraR. The DNA binding domain of TraR binds to tra box in the presence of 3OC8AHL molecules and then CMV minimal promoter will be activated by the activation domain of NF-κB p65 ^[3].

The DNA sequences of these genes are optimized for expressing in human cells considering the codon usage.

Characterization

In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atIPT4 and log1 genes to synthesize iP.

AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of AHLs, 3OC8HSL (C8) was chosen in the assay.

In order to achieve C8-dependent transcription in human cells, a chimeric transcription factor named RelA/NLS/TraR was constructed. This protein is comprised of the transcription activating domain of RelA (a kind of human NF-kB family), nuclear localization signal (NLS), and full-length TraR. This protein can bind to an appropriate enhancer sequence to activate transcription only in the presence of C8 (see below for details).

Isopentenyladenine (iP) is kind of a cytokinin, and we use it as a signal molecule from human to E. coli cells and for the inter-kingdom communication. 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. Surprisingly, 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 (See the AHK4 assay page).

Result

The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of atIPT4 and log1 was analyzed using quantitative RT-PCR. As shown in Figure 1, the CMV minimal promoter was activated following the addition of C8.

Figure 1: Result of the quantitative RT-PCR
The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. the concentrations of C8 used are indicated blow the bars.

Discussion

We confirmed that the transcription of atIPT4 and log1 genes were induced by C8 addition and the degree of induction depends on C8 concentration.

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.

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.

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.

Materials & Methods

Reference

[1] Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases, 2001. Kakimoto T.

[2] Direct control of shoot meristem activity by a cytokinin-activating enzyme, 2007. Kurakawa T. et. al

[3] A novel,inducible,eukaryotic gene expression system based on the quorum-sensing transcription factor TraR, 2003. Neddermann P. et. al