Human Cell Assay

Introduction

In this assay, we investigated whether human cells (EA.hy926 cell) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atlPT4 and log1 genes to synthesize iP.

Note

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

Summary

As shown in Fig.1, two kinds of constructs were introduced into EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses the chimeric protein, relA/NLS/traR. When relA/NLS/traR binds with C8, this complex binds to (tra box)7 sequence (the enhancer sequence; see below) and activates transcription from the CMV minimal promoter (CMV min). As a result, at the lPT4 and log1 genes are transcribed depending on C8. The atIPT4 and log1 gene products jointly work as IP synthetase.

Fig1. Construction of IP synthetase gene

Results

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Fig2. Result of the qualitative experiment

Discussion

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

Reference