Team:DTU Denmark/project

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.

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" and on the cloning part of making the biobricks in the notebook