Team:DTU Denmark/introduction private securkey Dhjg1mab2ak47

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<font size="4"><b>Theoretical background</b></font><br><br>
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<font size="4"><b>Genetic design</b></font><br><br>
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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>
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<p align="justify">The NAD<sup>+</sup>/NADH ratio sensor-protein Rex (<u>Re</u>do<u>x</u> regulator) has been discovered in the bacterium Streptomyces
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<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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coelicolor. In its host organism, the sensor works as a repressor and controls the gene expression of a large number of genes by recognizing and binding to a specific DNA-sequence termed ROP (<u>R</u>ex <u>OP</u>erator). NAD<sup>+</sup> and NADH compete for Rex binding, and the protein binds the ROP DNA-sequence only when NAD<sup>+</sup> is bound.</p><br>
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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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<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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<p align="justify"><i><b>Figure 1 - Gene design and redox regulation</b><br>
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<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 forming the Rexivator and will be transcribed constitutively leading to constant concentration of the sensor in the cell. The ROB sequence will be inserted into a specific promoter followed by a reporter gene, which will only be transcribed if 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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<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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<b><i>i) Reporter gene expression regulated by the Rexivator – an in vivo redox sensor</b></i><br>
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<p><b>Design specifications</b><b> </b><br />
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<p align="justify">The gene encoding green fluorescent protein (GFP) is widely used as a reporter gene in molecular biology. By placing the ROB promoter upstream of a GFP gene on a plasmid, and transforming the whole system into a yeast cell, GFP will be expressed at certain NAD<sup>+</sup>/NADH levels. When the Rexivator is bound to DNA, GFP expression will produce a visible and quantitatively measurable signal, which will be an indirect measure of the NAD<sup>+</sup>/NADH ratio.</p>
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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 is only expressed when REX-Activator is bound to ROB, which occurs at high NAD+/NADH levels.<b></b></p>
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[[Image:overallapproach.jpg|400px|thumb|center|The redox coupled system]]
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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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<p align="justify"><i><b>Figure 2 – Schematic overview of overall approach.</b><br>
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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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After the design, synthesis and transformation of the NAD<sup>+</sup>/NADH sensor, online measurement of reporter gene expression will be measured and oscillative behaviour of the productivity will be evaluated and used for further optimization.</i></p><br>
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<p align="justify">The measuring of cellular NAD<sup>+</sup>/NADH levels is usually a difficult process, especially due to fast changes in NAD<sup>+</sup>/NADH ratio that can occur when a sample is taken and exposed to slightly new conditions. With this sensor system, the quantitative measurement of the reporter gene will provide a fast and reliable way to determine the NAD<sup>+</sup>/NADH ratio in vivo. The plasmid can be transformed into a yeast strain allowing the NAD<sup>+</sup>/NADH ratio to be continuously monitored. As an example this plasmid can be used to study whether an engineered yeast strain has an altered NAD<sup>+</sup>/NADH ratio. The plasmid can be transformed into different yeast strains (e.g. wild type versus an engineered strain or two production strains) and the NAD<sup>+</sup>/NADH ratio can be compared.</p><br>
 
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<b><i>ii) Product formation regulated by the Rexivator – an attempt to improve and prolong chemostat processes</i></b><br>
 
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<p align="justify">When S. cerevisiae are grown continuously in a chemostat, the productivity of e.g. antibiotic or protein product gradually decreases [Personal correspondence with Novo Nordisk A/S]. This occurs during maximum production and is believed to be the result of metabolic adaption8 with reduced product formation as a consequence. The metabolic adaption is believed to occur because the cells are stressed by the extensive production. The cells will adapt to the new metabolic situation, which will gradually lead to lower production rates. This is highly undesirable in the biotech industry, as the chemostats will have to be restarted on a regular basis, which is costly and time consuming.</p>
 
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[[Image:oscillations.jpg|400px|thumb|center|The redox coupled system]]
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[[Image:dna sources.png|450px|thumb|center|<i><b>The genetic elements are from a variety of organisms.</b></i>]]
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<p align="justify"><i><b>Figure 3 – Chemostat fermentation with Rexivator system</b><br>
 
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Chemostat oscillations in the NAD+/NADH ratio as a result of the Yeast metabolic cycle (<b>A:</b> top graph) lead to change in the Rex DNA binding activity (<b>B:</b> middle graph) leading to controlled bursts in product of reporter gene expression (<b>C:</b> lower graph).</i></p><br>
 
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<p align="justify">A strategy to lower the effect of the metabolic adaption could be to couple the productivity with the yeast metabolic cycle (see grey box to the right). The NAD<sup>+</sup>/NADH poise oscillates in parallel with the yeast metabolic cycle. The synthesis of a given product will be put under control of the Rexivator, which will lead to periodic burst of gene expression and thus enzyme production (See figure 2). Consequently the cells will have time to recover periodically from the metabolic stress that occurs during the production phase. We believe that this will lead to a prolonged effective chemostat operation, and ultimately higher accumulation of product before a costly restart of the chemostat is required.<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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<p align="justify">By using computer modelling and experimental data from literature we will determine when in the metabolic cycle, it is favorable to produce, and when to let the cells rest. These predictions will be used to adjust and tune the sensing system, so that it initiates gene expression at appropriate times. The production over an extended period of time will then be monitored in a chemostat fermentation, to test if the yield has improved before restart is required.<p><br>
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<font size="3"><b>Yeast as a model organism for humans: Rexivator in cancer research</b></font><br>
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<p align="justify">Yeast is used as a model organism for humans in many research projects. As an example, studies of the yeast cell cycle have paved the way for most of the knowledge about the cell cycle in mammalian cells. As yeast has been widely used as a model organism for studying cancer an integrated tool for detecting a significant change in the redox potential would ease this research, especially because a rising NADH level is seen as a hallmark of carcinogenesis. The prospect for a cancer drug based on a DNA vaccine transcribed only at abnormally high NADH concentrations killing the cell could also be facilitated by research in this field.</p>
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<table border="1" cellspacing="0" cellpadding="0" align="center">
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    <tr>
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      <td width="141" valign="top"><p><b>Genetic element</b></td>
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      <td width="295" valign="top"><p><b>Required function</b></p></td>
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    </tr>
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      <td width="141" valign="top"><p>Constitutive promoter</p></td>
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      <td width="295" valign="top"><p>Constitutive expression of  REX-Activator fusion protein</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Kozak sequence</p></td>
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      <td width="295" valign="top"><p>Ribosome start-codon  recognition and enhanced initiating of translation</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>REX</p></td>
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      <td width="295" valign="top"><p>REX (<u>re</u>do<u>x</u> regulator) that binds to  ROP at high NAD+/NADH ratio </p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Linker</p></td>
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      <td width="295" valign="top"><p>To couple two protein  domains without disrupting their individual functions</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Activator domain</p></td>
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      <td width="295" valign="top"><p>Protein domain able to  activate transcription in eukaryotes in proximity of a minimal promoter.</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Nuclear Localization  Sequence (NLS)</p></td>
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      <td width="295" valign="top"><p>Translocation of the  REX-Activator protein to the nucleus</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Terminator 1</p></td>
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      <td width="295" valign="top"><p>Sequence that terminates  transcription</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>&nbsp;</p></td>
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      <td width="295" valign="top"><p>&nbsp;</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>ROB</p></td>
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      <td width="295" valign="top"><p>DNA sequence that REX binds  at high NAD+/NADH ratio</p></td>
 +
    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Minimal promoter</p></td>
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      <td width="295" valign="top"><p>Promoter devoid of  regulatory motifs. Only expression if an activator is bound upstream.</p></td>
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    </tr>
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    <tr>
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      <td width="141" valign="top"><p>GFP</p></td>
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      <td width="295" valign="top"><p>The amount of green  fluorescent protein can be quantitatively measured.</p></td>
 +
    </tr>
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    <tr>
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      <td width="141" valign="top"><p>Degradation signal</p></td>
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      <td width="295" valign="top"><p>Ensures fast degradation of  GFP</p></td>
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    </tr>
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    <tr>
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      <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 the DNA to loop out.</p></td>
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    </tr>
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  </table>
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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.

Comments or questions to the team? Please -- Comments of questions to webmaster? Please