We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s IGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. 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 undergo a conformational change that again will be indicated by 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 SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed.
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.
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