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Team:TMU-Tokyo/Project device1
From 2012.igem.org
Device1;
Sensing formaldehyde and then producing GFP
In frmRAB operon, which origin is chromosome of Escherichia coli, frmAB is normally repressed by frmR. But, when formaldehyde exists, frmR loses its inhibitory and frmAB is expressed.
This device is composed of frmR gene, its promoter and GFP gene.
By using this device, we can visualize formaldehyde with GFP. This visualization system enable to make us know how much of the formaldehyde concentration there is.
■Details of frmR effect
Christopher D. Herring and Frederick R. Blattner. 2004. "Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR" [PubMed]
■fig1.
a) When formaldehyde doesn't exist, frmR represses upstream promoter. In this state, transcription is stopped and GFP isn't produced.
b) When formaldehyde exists, it makes frmR lose activity. Then promoter is released from frmR's repress and begins to produce GFP.
Details of frmR repression
The sequence of frmR contains amber suppressor codon (UAG) at its 3' end. Amber suppressor codon recognizes its specialized tRNA. But the tRNA sometimes alters and gets capacity to carry amino acid. If amino acid comes, transcription is continued to more 7 amino acids and cording protein is degenerated. Wherein, frmR loses inhibitory.
Repressing ability of frmR was not shown molecularly but quantitatively. The downstream of frmR is inhibited by frmR and not by frmR added 7 amino acids.
Origin of frmR is guessed same as CsoR, copper-sensitive operon repressor. Both sequence is well preserved. It has been shown
that CsoR represses expression of genes located in downstream. So we guess it is same as frmR.
■Details of amber suppressor
G Eggertsson and D Söll. 1988. "Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli."[PMC]
■Function of frmR quantitative Analysis
Christopher D. Herring and Frederick R. Blattner. 2004. "Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR" [PubMed]
■fig2.(left)
Result of measuring expressed quantity of frmR, A, B by RT-PCR. Effects of frmR expression on levels of frmAB. It is clear that frmR can repress expression of downstream gene but frmR with 7 amino acid can't.
■fig3.(right)
