Chimeric Transcription Factor Assay

Introduction

In this assay, we checked whether human cells (EA.hy926 cell) receive AHL, a signaling molecule derived from E. coli and induce the transcription of atlPT4 gene and log1 gene which synthesize iP. Also, we checked whether IP (Isopentenyl adenine) molecules are synthesized in EA.hy926 cells by TLC.

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 3OC8AHL(C8) in the assay.

Summary

As shown in Fig. , we introduced two kinds of constructs into EA.hy926 cells by electroporation. CAG promoter constantly expresses relA/NLS/traR. After the protein binds with 3OC8AHL(C8), the complex exposes nuclear localization signal and activates CMVmin promoter in CMVmin_atIPT4_IVS_IRES_LOG1_polyA. As a result of pCMVmin's activation, downstream atlPT4 gene and log1 gene are transcripted. atlPT4 gene and log1 gene jointly work as IP synthetase.

Results

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Discussion

We checked the transcription of atlPT4 gene and log1 gene are induced by C8 addition and the induction depends on C8 concentration.

Reference

Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL 4: 159-165.