Team:DTU Denmark/introduction private securkey Dhjg1mab2ak47

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<font size="4"><b>Genetic design</b></font><br><br>
<font size="4"><b>Genetic design</b></font><br><br>
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<p>We have successfully designed and physically constructed a genetic system that couples the intracellular NAD+/NADH level to the gene expression of a reporter protein. The system has potentially many applications including in vivo online monitoring of the redox poise, production optimization and cancer research with yeast as a model organism (see <i>Applications</i>). We have demonstrated that the system functions as expected in <i>S. cerevisiae</i>.</p>
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<p>We have designed and physically constructed a genetic system that couples the intracellular NAD+/NADH level to the gene expression of a reporter protein. The system has potentially many applications including in vivo online monitoring of the redox poise, production optimization and cancer research with yeast as a model organism (see <i>Applications</i>).</p>
<p>The NAD+/NADH ratio is sensed by a system originating in <i>Streptomyces coellicolor</i>. In <i>S. coellicolor </i>the protein REX is a repressor and controls the gene expression of multiple genes by recognizing and binding to a specific DNA-sequence termed ROP (<u>R</u>ex <u>op</u>erator). NAD+ and NADH compete to associate with Rex, but only a REX:NAD+ association can bind the ROP DNA-sequence (Brekasis and Paget, 2003).</p>
<p>The NAD+/NADH ratio is sensed by a system originating in <i>Streptomyces coellicolor</i>. In <i>S. coellicolor </i>the protein REX is a repressor and controls the gene expression of multiple genes by recognizing and binding to a specific DNA-sequence termed ROP (<u>R</u>ex <u>op</u>erator). NAD+ and NADH compete to associate with Rex, but only a REX:NAD+ association can bind the ROP DNA-sequence (Brekasis and Paget, 2003).</p>
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<p>In <i>S. coellicolor</i> REX DNA binding represses expression of target genes, by physically hindering RNA-polymerases from binding the promoter. As the transcription machinery of eukaryotes is different and more complicated, there is no guarantee that repression will be effective in eukaryotes. REX has therefore been fused to a eukaryotic transcriptional activator, a widely used technique applied for the investigation of the GAL proteins and other systems (Sadowski et al. 1988). The REX-activator fusion-protein is able to bind the ROB sequence placed upstream of a minimal eukaryotic promoter that only supports transcription upon activation, and is devoid of regulatory motifs.</p>
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<p>In <i>S. coellicolor</i> REX DNA binding represses expression of target genes, by physically hindering RNA-polymerases from binding the promoter. As the transcription machinery of eukaryotes is different and more complicated, there are no guarantee that repression will be effective in eukaryotes. REX has therefore been fused to an eukaryotic transcriptional activator, a widely used technique applied for the investigation of the GAL proteins and other systems (Sadowski et al. 1988). The REX-activator fusion-protein is able to bind the ROB sequence placed upstream of a minimal eukaryotic promoter that only supports transcription upon activation. A certain NAD+/NADH ratio will activate the Redoxilator to recognize the ROB promoter, resulting in transcription of the reporter gene.</p>
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<p align="justify"><i><b>Gene design and redox regulation</b><br>
<p align="justify"><i><b>Gene design and redox regulation</b><br>
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<b>A:</b> The Rex gene will be fused to an activator domain and will be transcribed constitutively leading to constant concentration of the sensor in the cell. The ROB sequence and a minimal promoter is followed by a reporter gene, which will only be transcribed when the Rexivator complex is bound to the promoter. <b>B:</b> The Rexivator only binds the ROB DNA sequence under the condition of having NAD+ bound. Under these circumstances the fused activator domain summons the RNA polymerase and the reporter gene will be transcribed</i></p><br>
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<b>A:</b> The Rex gene has been fused to an activator domain and is transcribed constitutively, leading to constant concentration of the Rex-activator protein in the cell. The ROB sequence and a minimal promoter is followed by a reporter gene, which is only transcribed when the REX-activator fusion protein is bound to the promoter. <b>B:</b> The REX-activator only binds the ROB DNA sequence under the condition of having NAD+ bound. Under these circumstances the fused activator domain summons the RNA polymerase and the reporter gene is transcribed.</i></p><br>
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<font size="3"><b>Our synthetic biology project: The Redoxilator</b></font><br>
 
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<p align="justify">To achieve a system that senses changing levels in the NAD<sup>+</sup>/NADH ratio in the eukaryote <i>S. cerevisiae</i>, the gene encoding the Rex protein will be fused to a yeast activator domain, resulting in a new synthetic protein: the Redoxilator. The ROP sequence - the DNA binding site Rex can bind to - will be inserted into a yeast promoter, resulting in a promoter activated by the Redoxilator.</p>
 
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The Redoxilator system consists of two synthetic genes. One of the genes will be designed to code for a synthetic protein that activates transcription of the second gene only at a high NAD+/NADH ratio.
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<p><b>Design specifications</b><b> </b><br />
 +
The genetic system consists of two synthetic genes: one coding for the REX-activator fusion protein, and one coding for a yeast optimized GFP gene under control of ROB fused to a minimal promoter. With this system GFP is only expressed when REX-Activator is bound to ROB, which occurs at high NAD+/NADH levels.<b></b></p>
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<p align="justify">A certain NAD<sup>+</sup>/NADH ratio will activate the Redoxilator to recognize the ROB promoter resulting in transcription of a downstream gene. In this way the ROB promoter and the Redoxilator comprises the complete sensing system. The system can be coupled to the expression of virtually any gene of interest; making transcription solely dependent on the ratio of NAD<sup>+</sup>/NADH in the cell. In our iGEM project, the system will be used for two selected applications considered highly relevant: i) in vivo monitoring of NAD<sup>+</sup>/NADH in yeast, and ii) NAD<sup>+</sup>/NADH ratio regulated production of yeast products in chemostat processes.</p><br>
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</html>
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[[Image:redoxilator_biobrick_style.png|480px|thumb|center|<i><b>Simplified schematic representation of the synthetic genetic system on the DNA-level. The individual parts are described below.</b></i>]]
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<html>
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<p>The genetic elements and the requirements they need to fulfill are listed in the following table. The detailed description of the used genetic elements will not be made publicly available due to IP rights. The elements have been selected solely on their properties, and are from a variety of organisms - several of them are biobricks. All elements are codon optimized for <em>S. cerevisiae</em></p><br>
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<p><b>Design specifications</b><b> </b><br />
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The genetic system consists of two synthetic genes: one coding for the REX-activator fusion protein, and one coding for a yeast optimized GFP gene under control of ROB fused to a minimal promoter. With this system GFP will only be expressed when REX-Activator is bound to ROB, which occurs at high NAD+/NADH levels.<b></b></p>
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</html>
</html>
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[[Image:dnalevel_construct.png|500px|thumb|center|<i><b>Schematic representation of the synthetic genetic system on the DNA-level</b></i>]]
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[[Image:dna sources.png|450px|thumb|center|<i><b>The genetic elements are from a variety of organisms.</b></i>]]
<html>
<html>
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<p>The genetic elements and the requirements they need to fulfill are listed in the following table. The detailed description of the used genetic elements will not be made not publicly available due to IP rights.</p>
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<br>
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<p>Description and requirements of genetic elements.</p>
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<p align="center"><strong>Description and requirements of the genetic elements<br>constituting the Redoxilator device.</strong></p>
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      <td width="141" valign="top"><p><b>Genetic element</b></td>
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        <b>Genetic element</b></td>
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      <td width="295" valign="top"><p><b>Required function</b></p></td>
      <td width="295" valign="top"><p><b>Required function</b></p></td>
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      <td width="141" valign="top"><p>Terminator 1</p></td>
      <td width="141" valign="top"><p>Terminator 1</p></td>
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      <td width="295" valign="top"><p>Sequence that will terminate   transcription</p></td>
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      <td width="295" valign="top"><p>Sequence that terminates   transcription</p></td>
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      <td width="141" valign="top"><p>GFP</p></td>
      <td width="141" valign="top"><p>GFP</p></td>
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      <td width="295" valign="top"><p>The amount of green  fluprescent protein can be quantitatively measured.</p></td>
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      <td width="295" valign="top"><p>The amount of green  fluorescent protein can be quantitatively measured.</p></td>
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      <td width="141" valign="top"><p>Terminator 2</p></td>
      <td width="141" valign="top"><p>Terminator 2</p></td>
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      <td width="295" valign="top"><p>Terminates transcription.  Different from terminator 1 to avoid direct repeats, which can cause problems  with PCR-amplifications</p></td>
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      <td width="295" valign="top"><p>Terminates transcription.  Different from terminator 1 to avoid direct repeats, which can cause the DNA to loop out.</p></td>
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<p>
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<strong>References</strong><br>
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[Brekasis and Paget, 2003] Brekasis, D. and Paget, M. S. B. (2003). A novel sensor of nadh/nad+ redox poise in streptomyces coelicolor a3(2). EMBO J, 22(18):4856–4865.<br>
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[Sadowski et  al., 1988] Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988). Gal4-vp16 is an unusually potent transcriptional activator. Nature, 335(6190):563–564.
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  <b>The yeast metabolic cycle</b><br><br>
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<b>Synthetic Biology</b><br><br>
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<p align="left"><i>“Synthetic Biology is an art of engineering new biological systems that don’t exist in nature.”</i><br></p>
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<p align="right"><i>-Paras Chopra & Akhil Kamma</i><br><br></p>
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<p>In nature, biological molecules work together in complex systems to serve purposes of the cell. In synthetic biology these molecules are used as individual functional units that are combined to form tailored systems exhibiting complex dynamical behaviour. From ‘design specifications’ generated from computational modelling, engineering-based approaches enables the construction of such new specified gene-regulatory networks. The ultimate goal of synthetic biology is to construct systems that gain new functions, and the perspectives of the technology are enormous. It has already been used in several medical projects2 and is predicted to play a major role in biotech-production and environmental aspects.</p>
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<p>It has recently been shown by Tu <i>et al.</i> and Klevecz <i>et al.</i> that the expression of at least half of the genes monitored on a standard yeast gene chip will oscillate in a coordinated manner when grown under glucose limited conditions. The cells will shift between oxidative and reductive metabolism in a synchronized metabolic cycle with three phases: oxidative, reductive/building and reductive/
 
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charging. As oxygen will only be consumed in the oxidative phase, the dissolved oxygen will oscillate. Many metabolites and cofactors including NADH and NAD+ will also oscillate during this cycle as NADH is converted to NAD+ when oxygen is consumed.</p>
 
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Latest revision as of 21:44, 21 October 2009

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Theoretical background


The redoxilator

- Introduction
- Results
- Applications and perspectives


The USERTM assembly standard

- Principle
- Proof of concept
- Manual


USERTM fusion primer design software

- Abstract
- Instructions
- Output format

The project


Genetic design

We have designed and physically constructed a genetic system that couples the intracellular NAD+/NADH level to the gene expression of a reporter protein. The system has potentially many applications including in vivo online monitoring of the redox poise, production optimization and cancer research with yeast as a model organism (see Applications).

The NAD+/NADH ratio is sensed by a system originating in Streptomyces coellicolor. In S. coellicolor the protein REX is a repressor and controls the gene expression of multiple genes by recognizing and binding to a specific DNA-sequence termed ROP (Rex operator). NAD+ and NADH compete to associate with Rex, but only a REX:NAD+ association can bind the ROP DNA-sequence (Brekasis and Paget, 2003).

In S. coellicolor REX DNA binding represses expression of target genes, by physically hindering RNA-polymerases from binding the promoter. As the transcription machinery of eukaryotes is different and more complicated, there are no guarantee that repression will be effective in eukaryotes. REX has therefore been fused to an eukaryotic transcriptional activator, a widely used technique applied for the investigation of the GAL proteins and other systems (Sadowski et al. 1988). The REX-activator fusion-protein is able to bind the ROB sequence placed upstream of a minimal eukaryotic promoter that only supports transcription upon activation. A certain NAD+/NADH ratio will activate the Redoxilator to recognize the ROB promoter, resulting in transcription of the reporter gene.


The redox coupled system

The redox coupled system

Gene design and redox regulation
A: The Rex gene has been fused to an activator domain and is transcribed constitutively, leading to constant concentration of the Rex-activator protein in the cell. The ROB sequence and a minimal promoter is followed by a reporter gene, which is only transcribed when the REX-activator fusion protein is bound to the promoter. B: The REX-activator only binds the ROB DNA sequence under the condition of having NAD+ bound. Under these circumstances the fused activator domain summons the RNA polymerase and the reporter gene is transcribed.


Design specifications
The genetic system consists of two synthetic genes: one coding for the REX-activator fusion protein, and one coding for a yeast optimized GFP gene under control of ROB fused to a minimal promoter. With this system GFP is only expressed when REX-Activator is bound to ROB, which occurs at high NAD+/NADH levels.

Simplified schematic representation of the synthetic genetic system on the DNA-level. The individual parts are described below.

The genetic elements and the requirements they need to fulfill are listed in the following table. The detailed description of the used genetic elements will not be made publicly available due to IP rights. The elements have been selected solely on their properties, and are from a variety of organisms - several of them are biobricks. All elements are codon optimized for S. cerevisiae


The genetic elements are from a variety of organisms.


Description and requirements of the genetic elements
constituting the Redoxilator device.

Genetic element

Required function

Constitutive promoter

Constitutive expression of REX-Activator fusion protein

Kozak sequence

Ribosome start-codon recognition and enhanced initiating of translation

REX

REX (redox regulator) that binds to ROP at high NAD+/NADH ratio

Linker

To couple two protein domains without disrupting their individual functions

Activator domain

Protein domain able to activate transcription in eukaryotes in proximity of a minimal promoter.

Nuclear Localization Sequence (NLS)

Translocation of the REX-Activator protein to the nucleus

Terminator 1

Sequence that terminates transcription

 

 

ROB

DNA sequence that REX binds at high NAD+/NADH ratio

Minimal promoter

Promoter devoid of regulatory motifs. Only expression if an activator is bound upstream.

GFP

The amount of green fluorescent protein can be quantitatively measured.

Degradation signal

Ensures fast degradation of GFP

Terminator 2

Terminates transcription. Different from terminator 1 to avoid direct repeats, which can cause the DNA to loop out.


References
[Brekasis and Paget, 2003] Brekasis, D. and Paget, M. S. B. (2003). A novel sensor of nadh/nad+ redox poise in streptomyces coelicolor a3(2). EMBO J, 22(18):4856–4865.
[Sadowski et  al., 1988] Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988). Gal4-vp16 is an unusually potent transcriptional activator. Nature, 335(6190):563–564.


Synthetic Biology

“Synthetic Biology is an art of engineering new biological systems that don’t exist in nature.”

-Paras Chopra & Akhil Kamma

In nature, biological molecules work together in complex systems to serve purposes of the cell. In synthetic biology these molecules are used as individual functional units that are combined to form tailored systems exhibiting complex dynamical behaviour. From ‘design specifications’ generated from computational modelling, engineering-based approaches enables the construction of such new specified gene-regulatory networks. The ultimate goal of synthetic biology is to construct systems that gain new functions, and the perspectives of the technology are enormous. It has already been used in several medical projects2 and is predicted to play a major role in biotech-production and environmental aspects.

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