Team:DTU Denmark/project

The project is divided into four main sections that can be accessed to the left. Each section is briefly introduced below.

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. Expected future applications of the system is heterologous redox coupled protein production in yeast, and a possible application in cancer research

In this part of the project we propose a new parts-assembly standard for Biobricks based on USER cloning. The USER fusion assembly standard allows rapid construction of multi-part devices, without some of the drawbacks of the restriction-enzyme based standard biobrick assembly method.

USER fusion primer design software
When designing constructs with more than two biobricks using USER fusion, several parameters have to be taken into account when designing primers needed for the fusion. For this project we have programmed an online tool, that can automatically find the optimal primers for the fusion of up to 9 biobricks at once while taking several parameters into account including melting temperature, primer length and placement as well as avoidance of nearly identical primers.

Experimental results
In this section the results of our experimental work is described including:

• Construction and characterization of the biobricks
  
• Experiments and results from testing the Redoxilator.
  
• Demonstration of the USER fusion assembly standard.

Biobricks designed and submitted

Besides the main project, four novel biobricks were made as a complement of our project and as a useful tool for the construction of future devices (see "parts submitted" ):

• A GFP variant optimized for expression in yeast.
  
• A protein destabilization sequence, which allows rapid protein turnover when appended to any protein.
  
• 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.
  
• A USER cassette that will allow insertion of PCR fragments using the novel USER biobrick assembly standard.