Team:DTU Denmark/theory

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.

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 redox coupled system

The redox coupled system

Figure 1 - Gene design and redox regulation
A: 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: 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

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.