Team:TMU-Tokyo/Project device1

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In <i>frmRAB</i> operon, which origin is chromosome of <i>Escherichia coli</i>, <i>frmAB</i> is normally repressed by frmR. But, when formaldehyde exists, <i>frmR</i> loses its inhibitory(2). This device is composed of <i>frmR</i> gene, its promoter and GFP gene.</p><Br>
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In <i>frmRAB</i> operon, which origin is chromosome of <i>Escherichia coli</i>, <i>frmAB</i> expression is normally repressed by FrmR. But, when formaldehyde exists, FrmR loses its inhibitory and <i>frmAB</i> is expressed.  
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This device is composed of <i>frmR</i> gene, its promoter and GFP gene.</p>
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<center><Img Src="https://static.igem.org/mediawiki/2012/3/3a/Device1.png"></center><Br>
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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.<Br>
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2. <Strong>Christopher D. Herring and Frederick R. Blattner.</Strong> 2004. Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator <i>frmR</i>[<A Href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC522192/">PubMed</A>]</p>
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<B>■Details of <i>frmR</i> effect</B>
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<Br>Christopher D. Herring and Frederick R. Blattner. 2004. "Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator <i>frmR</i>" [<A Href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC522192/">PubMed</A>]<Br>
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<b>■fig1.<Br>
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a)</b> When formaldehyde doesn't exist, <i>frmR</i> represses upstream promoter. In this state, transcription is stopped and GFP isn't produced.<Br>
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<b>b)</b> When formaldehyde exists, it makes <i>frmR</i> lose activity. Then promoter is released from FrmR's repress and begins to produce GFP.<Br>
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<Br><p class="description"><Strong>Details of <i>frmR</i> repression</Strong><Br>
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The sequence of <i>frmR</i> 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, <i>frmR</i> loses inhibitory.<Br>
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Repressing ability of <i>frmR</i> was not shown molecularly but quantitatively. The downstream of frmR is inhibited by frmR and not by frmR added 7 amino acids.<Br>
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<center><p class="description"><Img Src="https://static.igem.org/mediawiki/2012/6/6c/FrmR.jpg" Width="53%" Height="53%">
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<Img Src="https://static.igem.org/mediawiki/2012/f/ff/FrmR2.jpg" Width="44% Height="44%" ><Br>
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Origin of FrmR is guessed same as RcnR, nickel and cobalt-sensitive operon repressor. Both sequence is well preserved. It has been shown that RcnR represses expression of genes located in downstream by coordination of DNA-binding and metal-binding activities. So we guess <i>frmR</i> has same system.<Br>
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<b>■Details of amber suppressor</b><Br>
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G Eggertsson and D Söll. 1988. "Transfer ribonucleic acid-mediated suppression of termination codons in <i>Escherichia coli</i>."[<A Href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373150/">PMC</A>]<Br>
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<B>■Function of <i>frmR</i> quantitative Analysis</B><Br>
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Christopher D. Herring and Frederick R. Blattner. 2004. "Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator <i>frmR</i>" [<A Href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC522192/">PubMed</A>]<Br>
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<B>■fig2.(left)</B><Br>
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Result of measuring expressed quantity of <i>frmR, A, B</i> by RT-PCR. It is clear that <i>frmR</i> can repress expression of downstream gene but <i>frmR</i> with 7 amino acid can't. <Br>
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<B>■fig3.(right)</B><Br>
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Result of measuring expressed quantity of <i>frmR, A, B</i> by RT-PCR. Formaldehyde surely activates <i>frmRAB</i> expression.<Br>
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Each figs were qupted from the paper of ■Function of frmR quantitative Analysis
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<B>■Domain family; RcnR-FrmR-like_DUF156</B><Br>
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Transcriptional regulators RcnR and FrmR, and related domains; this domain family was previously known as part of DUF156.<Br>
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[<A Href="http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?hslf=1&uid=cd10153&#seqhrch">NCBI</A>]<Br>
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<B>■<i>RcnR</i> repression system</B><Br>
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Jeffrey S. Iwig, Peter T. Chivers. 2009. "DNA recognition and wrapping by Escherichia coli RcnR" <Br>
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[<A Href="http://www.ncbi.nlm.nih.gov/pubmed/19703465?dopt=Abstract">PubMed</A>]
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<A Href="Project">Project Top</A>  <A Href="Project_abstract">Abstract</A>  Device1  <A Href="Project_device2">Device2</A>  <A Href="Project_device3">Device3</A><Br><Br>
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Latest revision as of 16:24, 23 September 2012

 


Device1;

Sensing formaldehyde and then producing GFP


 
 

In frmRAB operon, which origin is chromosome of Escherichia coli, frmAB expression 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 RcnR, nickel and cobalt-sensitive operon repressor. Both sequence is well preserved. It has been shown that RcnR represses expression of genes located in downstream by coordination of DNA-binding and metal-binding activities. So we guess frmR has same system.




■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. It is clear that frmR can repress expression of downstream gene but frmR with 7 amino acid can't.
■fig3.(right)
Result of measuring expressed quantity of frmR, A, B by RT-PCR. Formaldehyde surely activates frmRAB expression.

Each figs were qupted from the paper of ■Function of frmR quantitative Analysis







■Domain family; RcnR-FrmR-like_DUF156
Transcriptional regulators RcnR and FrmR, and related domains; this domain family was previously known as part of DUF156.
[NCBI]

■RcnR repression system
Jeffrey S. Iwig, Peter T. Chivers. 2009. "DNA recognition and wrapping by Escherichia coli RcnR"
[PubMed]

 
 


Project Top Abstract Device1 Device2 Device3

 

Retrieved from "http://2012.igem.org/Team:TMU-Tokyo/Project_device1"