Team:DTU Denmark/introduction private securkey Dhjg1mab2ak47

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.

The redox coupled system

The redox coupled system

Gene design and redox regulation
A: 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: 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

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.

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.

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 will only be expressed when REX-Activator is bound to ROB, which occurs at high NAD+/NADH levels.

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.