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

The USER assembly standard

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.

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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