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Team:DTU Denmark/theory
From 2009.igem.org
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placing the ROB promoter upstream of a GFP gene on a plasmid, and transforming the whole system into a yeast | 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 | 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. | + | will produce a visible and quantitatively measurable signal, which will be an indirect measure of the NAD<sup>+</sup>/NADH ratio.<br> |
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<p align="justify"><i><b>Figure 2 – Schematic overview of overall approach.</b><br> | <p align="justify"><i><b>Figure 2 – Schematic overview of overall approach.</b><br> | ||
| - | 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. | + | 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.<br><br> |
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| + | 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.<br><br> | ||
Revision as of 19:36, 8 October 2009
| Home | The Team | The Project | Parts submitted | Modelling | Notebook |
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The redoxilator - Theoretical background - Yeast as a model organism - Practical approach The USER assembly standard - Principle - Proof of concept - Manual - Primer design software |
The project Theoretical background The NAD+/NADH ratio sensor-protein Rex (Redox regulator) has been discovered in the bacterium Streptomyces 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 (Rex OPerator). NAD+ and NADH compete for Rex binding, and the protein binds the ROP DNA-sequence only when NAD+ is bound. Our synthetic biology project: The Redoxilator To achieve a system that senses changing levels in the NAD+/NADH ratio in the eukaryote S. cerevisiae, 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. 
Figure 1 - Gene design and redox regulation
A certain NAD+/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+/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+/NADH in yeast, and ii) NAD+/NADH ratio regulated production of yeast products in chemostat processes. i) Reporter gene expression regulated by the Rexivator – an in vivo redox sensorThe 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+/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+/NADH ratio. 
Figure 2 – Schematic overview of overall approach. |
The yeast metabolic cycle It has recently been shown by Tu et al. and Klevecz et al. 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/ 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. |
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