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From 2010.igem.org

The aim of the project is to present a contribution to the IGEM competition in synthetic biology. The contribution consists of creating a sensor for cellular stress readouts. The organism of choice is yeast and the protein that is used to study the phenomena is SNF1.

The project is executed through two main experimental pathways. Both experimental setups will utilize FRET to visualize the conformational change that is the result of the activation of the SNF1 protein. The first approach consists of creating a fusion protein consisting of the SNF1 protein and two fluorescent proteins, namely EYFP and ECFP. The idea is that when the protein is activated it undergoes a conformational change and a FRET-signal will be visible. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and will undergo a conformational change that will be visible due to the fluorescent tags.

To reach the goal several steps need to be performed. Firstly the most suitable positions of the fluorescent proteins need to be determined. Subsequently the fusion proteins are created through fusion PCR. The fusion proteins are then put into vectors by the means of homologous recombination after which they are transformed into yeast. The plasmids are then purified and transformed into E. coli to amplify the yield, sequence the insert and select the plasmids carrying the correct insert. The last step is to transform a new yeast strain with the plasmids, expose the cells to different stress factors and study the FRET signal.

The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient and easily finding out at which concentration or substance that the cells are stressed.