Team:DTU Denmark/genetic design

We have 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).

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 are 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.

The redox coupled system

The redox coupled system

Gene design and redox regulation
A: The Rex gene has been fused to an activator domain and is transcribed constitutively, leading to constant concentration of the Rex-activator protein in the cell. The ROB sequence and a minimal promoter is followed by a reporter gene, which is only be transcribed when the REX-activator fusion protein is bound to the promoter. B: The REX-activator 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 is transcribed

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

Simplified schematic representation of the synthetic genetic system on the DNA-level. The individual parts are described below.

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 publicly available due to IP rights. The elements have been selected solely on their properties, and are from a variety of organisms - several of them are biobricks. All elements are codon optimized for S. cerevisiae

The genetic elements are from a variety of organisms.

Modelling
To guide the design process and to simulate how our genetic device would operate in vivo, we performed thorough mathematical modelling of the relation between the input and output of our system (the detailed model is available here). The model predicts how the GFP-concentration in a transformed cell relates to the internal NAD+/NADH level (indicative of the redox state).
The model takes the following into account:

i) transcription factor activation,
ii) promoter activation,
iii) synthesis and degradation of mRNA and finally,
iv) synthesis, dilution and degradation of GFP.

The model provided understanding of the influence of the various parameters on the behavior of the system. The most important discovery, was the fact that due to the relatively slow degradation of GFP, the system would have a slow response time: a change in NAD+/NADH level would not be reflected fast enough for the system to be efficient.
The solution to our problem was to bring down the half-life of the GFP. This was done by the fusion of GFP and a PEST degradation signal from the yeast protein Cln2, which has been demonstrated to reduce the half-life from 7 hours to 30 minutes (Mateus and Avery, 2000). The fast degradable GFP-Cln2 has been created as a biobrick and submitted to the registry (available here)

Design and submission of three novel biobricks
The possibility of making a fast degradable version of specific proteins is essential when engineering biological systems by a synthetic biology approach. To bring this possibility to other registry users, we have designed a biobrick that allows fast degradation of any protein in S. cerevisiae, in a simple and easy way: the biobricks are assembled by any standard that allows protein-fusions.
Biobricks

Description and requirements of the genetic elements
constituting the Redoxilator device.

References
[Brekasis and Paget, 2003] Brekasis, D. and Paget, M. S. B. (2003). A novel sensor of nadh/nad+ redox poise in streptomyces coelicolor a3(2). EMBO J, 22(18):4856–4865.
[Mateus and Avery, 2000] Mateus, C. and Avery, S. V. (2000). Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. Yeast, 16(14):1313–1323.
[Sadowski et al., 1988] Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988). Gal4-vp16 is an unusually potent transcriptional activator. Nature, 335(6190):563–564.