Team:TMU-Tokyo/Project device2

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<i>Escherichia coli</i> has original formaldehyde dehydrogenase, but it depends on glutathione (existence). Instead of it, we found glutathione-independent formaldehyde dehydrogenase in <i>Pseudomonas putida</i>. This device is composed of its gene and constitutive promoter.<Br></p>
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This device is composed of formaldehyde dehydrogenase in <i>Pseudomonas putida</i> (PFDH) gene and the strongest constitutive promoter (<A Href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</A>), the srtongest RBS (<A Href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</A>). The reason we don't use repressor like <i>frmR</i> is to increase resistance of formaldehyde. We use the same strategy in <A Href="https://2012.igem.org/Team:TMU-Tokyo/Project_device3">device3</A> to increase resistance of formate.<Br>
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<i>Escherichia coli</i> has original formaldehyde dehydrogenase (FDH), but it depends on glutathione existence. Instead of it, we found glutathione-independent FDH in <i>Pseudomonas putida</i> (PFDH). Formaldehyde dehydrogenase changes formaldehyde to formate. (HCHO → HCOOH)<Br>
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The reason we didn't select <i>Escherichia coli</i>'s own FDH is that glutathione-independent one can act in much more situations. For example, if Cheff Ant E.coli dies, <i>Escherichia coli</i>'s own FDH loses its substrate because of glutathione absence. FDH and PFDH are homologous and their sequences are well preserved.<Br>
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In additon to the glutathione-independence, PFDH can be increased its activity by a few amino acids mutation. It was measured that the activity increased 1.7 times stronger.<Br>
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■<b><i>E. coli</i> original FDH</b><Br>
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Enzyme: formaldehyde dehydrogenase, glutathione-dependent<Br>
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[<A Href="http://ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME-IN-RXN-DISPLAY&object=ADHC-CPLX">EcoCyc</A>]
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■<b>PFDH enzyme activity measuring</b><Br>
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Ando M, Yoshimoto T, Ogushi S, Rikitake K, Shibata S, Tsuru D. 1979. "Formaldehyde dehydrogenase from <i>Pseudomonas putida</i>. Purification and some properties"<Br>
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[<A Href="http://www.ncbi.nlm.nih.gov/pubmed/571868">PubMed</A>]<Br>
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■<b>PFDH can be stronger by amino acids mutation</b><Br>
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Fujii Y, Yamasaki Y, Matsumoto M, Nishida H, Hada M, Ohkubo K. 2004. "The Artificial Evolution of an Enzyme by Random Mutagenesis: The Development of Formaldehyde Dehydrogenase"<Br>
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<A Href="Project">Project Top</A>  <A Href="Project_abstract">Abstract</A>  <A Href="Project_device1">Device1</A>  Device2  <A Href="Project_device3">Device3</A><Br><Br>
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Latest revision as of 01:55, 27 September 2012

 


Device2;

Producing formaldehyde dehydrogenase, changing formaldehyde to formate


 
 

This device is composed of formaldehyde dehydrogenase in Pseudomonas putida (PFDH) gene and the strongest constitutive promoter (BBa_J23100), the srtongest RBS (BBa_B0034). The reason we don't use repressor like frmR is to increase resistance of formaldehyde. We use the same strategy in device3 to increase resistance of formate.

Escherichia coli has original formaldehyde dehydrogenase (FDH), but it depends on glutathione existence. Instead of it, we found glutathione-independent FDH in Pseudomonas putida (PFDH). Formaldehyde dehydrogenase changes formaldehyde to formate. (HCHO → HCOOH)

The reason we didn't select Escherichia coli's own FDH is that glutathione-independent one can act in much more situations. For example, if Cheff Ant E.coli dies, Escherichia coli's own FDH loses its substrate because of glutathione absence. FDH and PFDH are homologous and their sequences are well preserved.


In additon to the glutathione-independence, PFDH can be increased its activity by a few amino acids mutation. It was measured that the activity increased 1.7 times stronger.










■E. coli original FDH
Enzyme: formaldehyde dehydrogenase, glutathione-dependent
[EcoCyc]


■PFDH enzyme activity measuring
Ando M, Yoshimoto T, Ogushi S, Rikitake K, Shibata S, Tsuru D. 1979. "Formaldehyde dehydrogenase from Pseudomonas putida. Purification and some properties"
[PubMed]

■PFDH can be stronger by amino acids mutation
Fujii Y, Yamasaki Y, Matsumoto M, Nishida H, Hada M, Ohkubo K. 2004. "The Artificial Evolution of an Enzyme by Random Mutagenesis: The Development of Formaldehyde Dehydrogenase"
[J-Stage]


 

Project Top Abstract Device1 Device2 Device3



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