Team:DTU Denmark/introduction private securkey Dhjg1mab2ak47

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Theoretical background


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.


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


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.


Genetic element

Required function

Constitutive promoter

Constitutive expression of REX-Activator fusion protein

Kozak sequence

Ribosome start-codon recognition and enhanced initiating of translation

REX

REX (redox regulator) that binds to ROP at high NAD+/NADH ratio

Linker

To couple two protein domains without disrupting their individual functions

Activator domain

Protein domain able to activate transcription in eukaryotes in proximity of a minimal promoter.

Nuclear Localization Sequence (NLS)

Translocation of the REX-Activator protein to the nucleus

Terminator 1

Sequence that will terminate transcription

 

 

ROB

DNA sequence that REX binds at high NAD+/NADH ratio

Minimal promoter

Promoter devoid of regulatory motifs. Only expression if an activator is bound upstream.

GFP

The amount of green fluprescent protein can be quantitatively measured.

Degradation signal

Ensures fast degradation of GFP

Terminator 2

Terminates transcription. Different from terminator 1 to avoid direct repeats, which can cause problems with PCR-amplifications



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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