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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 Theoretical background Genetic design and results 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 Applications). We have demonstrated that the system functions as expected in S. cerevisiae. 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 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. FIGURE REX-Activator: mechanism of action. Low NAD+/NADH levels result in no DNA-binding of REX-activator and thus no expression of the GFP gene. A high NAD+/NADH level results in activated REX-Activator protein that binds to ROB, leading to transcription of the GFP gene placed downstream. Design specifications FIGURE Schematic representation of the synthetic genetic system on the DNA-level. 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. Description and requirements of genetic elements.
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. 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. The
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 sensor 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+/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 measuring of cellular NAD+/NADH levels is usually a difficult process, especially due to fast changes in NAD+/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+/NADH ratio in vivo. The plasmid can be transformed into a yeast strain allowing the NAD+/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+/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+/NADH ratio can be compared. ii) Product formation regulated by the Rexivator – an attempt to improve and prolong chemostat processes 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. 
Figure 3 – Chemostat fermentation with Rexivator system 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+/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.
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.
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. |
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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