Team:DTU Denmark/parts

From 2009.igem.org

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<font size="3"><b>cln2 PEST destabilization domain for rapid protein turnover</b></font><br>
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<font size="3"><b>A protein destabilization sequence for yeast</b></font><br>
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<p align="justify">C-terminal domain of Saccharomyces cerevisiae cyclin 2 (CLN2) gene. It has been shown that this region of the protein, which is rich of PEST motifs, leads to a destabilization of the protein. Hence this Tag can be used to increase the turn-over rate of a protein. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues changes the half-life of a GFP from 7h and down to 30 minutes.</p>
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<p align="justify"><i>This biobrick can be fused to any protein of interest and enables careful control of protein level by increasing degradation rate.</i></p>
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<p align="justify">This destabilization sequence for rapid protein turnover consists on the C-terminal domain of <i>Saccharomyces cerevisiae</i> cyclin 2 (CLN2) gene. It has been shown that this region of the protein, which is rich in PEST motifs, leads to a destabilization of a protein. Hence this Tag can be used to increase the turn-over rate of a protein. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues changes the half-life of a GFP from 7h and down to 30 minutes.</p>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194000" target="_blank">BBa_K194000</a>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194000" target="_blank">BBa_K194000</a>
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<font size="3"><b>GFP (a yeast- and FACS-optimized GFP variant)</b></font><br>
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<font size="3"><b>Yeast optimized Green Fluorescent Protein (yGFP)</b></font><br>
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<p align="justify">Green Fluorescent Protein (GFP) codon optimized for yeast. </p>
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<p align="justify"><i>Now the parts registry also offers a GFP for yeast - codon optimized and enhanced. By expanding the host organism, this is an improvement of an existing biobrick: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_E0040" target="_blank">BBa_E0040</a>
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</i></p>
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<p align="justify"> This constitutively expressed GFP, originally from <i>Aequorea victoria</i> was codon optimized and developed as a reporter for gene expression in <i>Saccharomyces cerevisiae</i> according to Cormak et al (1997). In addition, two amino acid changes were included enabling a far more bright florescence compared to the wild type in <i>E.coli</i>.
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Maximum absorbance: 490 nm.</p>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194001" target="_blank">BBa_K194001</a>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194001" target="_blank">BBa_K194001</a>
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<font size="3"><b>Destabilized GFP for yeast</b></font><br>
<font size="3"><b>Destabilized GFP for yeast</b></font><br>
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<p align="justify">This variant of biobrick BBa_K194001 has been destabilized by fusing biobrick BBa_K194000. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues of the Cln2 PEST signal, changes the half-life of a GFP from 7h and down to 30 minutes.</p>
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<p align="justify"><i> With a degradation half life of only 30 min, this is a fast reporter signal for yeast. It is constructed by the combination of the two above described biobricks (yGFP and Cln2 PEST signal), and is thus a further improvement of the existing biobrick for GFP:<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_E0040" target="_blank">BBa_E0040</a>
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</i></p>
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<p align="justify">A fast degradable GFP that has a half-life of 30 min. This variant of biobrick BBa_K194001 has been destabilized by fusing it with biobrick BBa_K194000. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues of the Cln2 PEST signal, changes the half-life of a GFP from 7h down to 30 minutes.</p>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194002" target="_blank">BBa_K194002</a>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194002" target="_blank">BBa_K194002</a>
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<font size="3"><b>USER cassette for insertion of USER fragments</b></font><br>
<font size="3"><b>USER cassette for insertion of USER fragments</b></font><br>
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<p align="justify">This biobrick contains a PacI/Nt.BbvCI USER cassette for insertion of PCR fragments using the USER cloning standard. PCR fragments containing uracil is treated with a uracil DNA glycosylase that removes the uracil exposing a predesigned 8bp overhang allowing for cloning without the need for ligase. </p>
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<p align="justify"><i> USER cloning allows fast and efficient cloning of PCR fragments into plasmids. Here we present the first USER cassette for the purpose of USER cloning.
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</i></p>
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<p align="justify">This biobrick will allow insertion of PCR fragments using the novel USERTM biobrick assembly standard (<a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_39:_The_USER_cloning_standard">BBF RFC 39</a>). It contains a PacI/Nt.BbvCI USER cassette for insertion of PCR fragments using the USER cloning standard. PCR fragments containing uracil is treated with a uracil DNA glycosylase that removes the uracil exposing a predesigned 8bp overhang allowing for cloning without the need for ligase </p>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194003" target="_blank">BBa_K194003</a>
Registry id: <a href="http://partsregistry.org/Part:BBa_K194003" target="_blank">BBa_K194003</a>
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<b>References</b><br>
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Mateus and Avery, 2000. Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. Yeast 2000; 16: 1313±1323.
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The Registry is based on the principle of "get some, give some". Registry users benefit from using the parts and information available from the Registry in designing their engineered biological systems. In exchange, the expectation is that Registry users will, in turn, contribute back information and data on existing parts and new parts that they make to grow and improve this community resource.</p>
The Registry is based on the principle of "get some, give some". Registry users benefit from using the parts and information available from the Registry in designing their engineered biological systems. In exchange, the expectation is that Registry users will, in turn, contribute back information and data on existing parts and new parts that they make to grow and improve this community resource.</p>
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Latest revision as of 03:03, 22 October 2009

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Welcome to the DTU iGEM wiki!


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A protein destabilization sequence for yeast

This biobrick can be fused to any protein of interest and enables careful control of protein level by increasing degradation rate.

This destabilization sequence for rapid protein turnover consists on the C-terminal domain of Saccharomyces cerevisiae cyclin 2 (CLN2) gene. It has been shown that this region of the protein, which is rich in PEST motifs, leads to a destabilization of a protein. Hence this Tag can be used to increase the turn-over rate of a protein. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues changes the half-life of a GFP from 7h and down to 30 minutes.

Registry id: BBa_K194000

Yeast optimized Green Fluorescent Protein (yGFP)

Now the parts registry also offers a GFP for yeast - codon optimized and enhanced. By expanding the host organism, this is an improvement of an existing biobrick: BBa_E0040

This constitutively expressed GFP, originally from Aequorea victoria was codon optimized and developed as a reporter for gene expression in Saccharomyces cerevisiae according to Cormak et al (1997). In addition, two amino acid changes were included enabling a far more bright florescence compared to the wild type in E.coli. Maximum absorbance: 490 nm.

Registry id: BBa_K194001

Destabilized GFP for yeast

With a degradation half life of only 30 min, this is a fast reporter signal for yeast. It is constructed by the combination of the two above described biobricks (yGFP and Cln2 PEST signal), and is thus a further improvement of the existing biobrick for GFP:BBa_E0040

A fast degradable GFP that has a half-life of 30 min. This variant of biobrick BBa_K194001 has been destabilized by fusing it with biobrick BBa_K194000. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues of the Cln2 PEST signal, changes the half-life of a GFP from 7h down to 30 minutes.

Registry id: BBa_K194002

USER cassette for insertion of USER fragments

USER cloning allows fast and efficient cloning of PCR fragments into plasmids. Here we present the first USER cassette for the purpose of USER cloning.

This biobrick will allow insertion of PCR fragments using the novel USERTM biobrick assembly standard (BBF RFC 39). It contains a PacI/Nt.BbvCI USER cassette for insertion of PCR fragments using the USER cloning standard. PCR fragments containing uracil is treated with a uracil DNA glycosylase that removes the uracil exposing a predesigned 8bp overhang allowing for cloning without the need for ligase

Registry id: BBa_K194003

References
Mateus and Avery, 2000. Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. Yeast 2000; 16: 1313±1323.
The Registry of Standard Biological Parts

The Registry is a collection of ~3200 genetic parts that can be mixed and matched to build synthetic biology devices and systems. Founded in 2003 at MIT, the Registry is part of the Synthetic Biology community's efforts to make biology easier to engineer. It provides a resource of available genetic parts to iGEM teams and academic labs.

The Registry is based on the principle of "get some, give some". Registry users benefit from using the parts and information available from the Registry in designing their engineered biological systems. In exchange, the expectation is that Registry users will, in turn, contribute back information and data on existing parts and new parts that they make to grow and improve this community resource.


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