Team:DTU Denmark/project

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The project

Notebook

The redoxilator

- Genetic design
- Applications and perspectives
- Results
- Safety considerations

The USER assembly standard

- USER fusion of biobricks

USER fusion primer design software

- Abstract
- Instructions
- Output format

The project

Project abstract

The Redoxilator

By in silico design and computer modelling followed by gene synthesis, we have constructed a molecular NAD/NADH ratio sensing system in Saccharomyces cerevisiae. The sensor works as an inducible transcription factor being active only at certain levels of the NAD/NADH ratios. By the coupling of a yeast optimized fast degradable GFP, the system can be used for in vivo monitoring of NAD/NADH redox poise. A future novel application of the system is heterologous redox coupled protein production in yeast.

The redox coupled system

The USER fusion standard

Another part of our project is the proposal of a new parts-assembly standard for Biobricks based on USER cloning. With this technique, not based on restriction enzymes, all parts independent of function can be assembled without leaving any scars from the restriction enzyme digestions.

Biobricks designed and submitted

Besides the main project, 4 biobricks were made as a complement of our project and as a useful tool for the construction of future devices
1) A GFP variant optimized for expression in yeast
2) A protein destabilization sequence, which allows rapid protein turnover when appended to any protein
3) A device made from 1+2 as a proof of concept and very useful biobrick in itself: A fast degradable GFP that has a halflife of 30 min. compared to 7 hours without destabilization.
4) A USER cassette that will allow insertion of PCR fragments using the novel USER biobrick assembly standard.
More details about our biobricks under "parts submitted"

Achievements

Achievements

Redox sensing device
Two novel genes have been designed and synthesized each comprised of 5 genetic elements. Together they function as a device termed the Redoxilator that can sense the internal redox state of a yeast cell, and output a reporter signal. Extensive mathematical modelling was performed to simulate how the construct would operate in vivo.

Biobricks
DNA of several new biobricks have been designed and submitted including a yeast optimized GFP reporter protein, a protein degradation sequence and a fast degradable yeast GFP. (Bronze medal)

We have demonstrated that our USER fusion biobrick works as expected and documented it (silver medal)

USER fusion Assembly standard
A new biobrick assembly standard that allows the rapid construction of multi-part devices have been developed and documented. The assembly standard offers many benefits: All restriction sites are allowed, multiple biobricks can be joined in one step, the result is scar-free making it ideal for protein fusions and more. (Gold medal)

USER-fusion primer design software
A novel and very useful software tool have been developed that can automatically design the optimal primers for USER fusion assembly of 2-9 biobricks, taking several parameters into account.

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