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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 Genetic design 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. A certain NAD+/NADH ratio will activate the Redoxilator to recognize the ROB promoter resulting in transcription of the reporter gene. 
Gene design and redox regulation Design specifications 
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.
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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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